Recombinant microalgae including sucrose invertase and thioesterase

ABSTRACT

Disclosed herein are methods of manufacturing renewable chemicals through the manufacture of novel triglyceride oils followed by chemical modification of the oils. Methods such as transesterification, hydrogenation, hydrocracking, deoxygenation, isomerization, interesterification, hydroxylation, hydrolysis and saponification are disclosed. Novel oils containing fatty acid chain lengths of C8, C10, C12 or C14 are also disclosed and are useful as feedstocks in the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/555,009, filed Jul. 20, 2012, now U.S. Pat. No. 9,062,294, which is acontinuation of U.S. application Ser. No. 13/550,412, filed Jul. 16,2012, now U.S. Pat. No. 8,435,767, which is a continuation of U.S.application Ser. No. 12/981,409, filed Dec. 29, 2010, now U.S. Pat. No.8,222,010, which is a continuation of U.S. application Ser. No.12/628,149, filed Nov. 30, 2009, now U.S. Pat. No. 7,883,882, whichclaims the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication No. 61/118,590, filed Nov. 28, 2008, U.S. Provisional PatentApplication No. 61/118,994, filed Dec. 1, 2008, U.S. Provisional PatentApplication No. 61/174,357, filed Apr. 30, 2009, and U.S. ProvisionalPatent Application No. 61/219,525, filed Jun. 23, 2009. Each of theseapplications is incorporated herein by reference in its entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“458558-Sequence.txt”, created on Feb. 19, 2015 and containing 348,478bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to the production of oils, fuels, andoleochemicals made from microorganisms. In particular, the disclosurerelates to oil-bearing microalgae, methods of cultivating them for theproduction of useful compounds, including lipids, fatty acid esters,fatty acids, aldehydes, alcohols, and alkanes, and methods and reagentsfor genetically altering them to improve production efficiency and alterthe type and composition of the oils produced by them.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for buried combustible geologic depositsof organic materials, formed from decayed plants and animals that havebeen converted to crude oil, coal, natural gas, or heavy oils byexposure to heat and pressure in the earth's crust over hundreds ofmillions of years. Fossil fuels are a finite, non-renewable resource.

Increased demand for energy by the global economy has also placedincreasing pressure on the cost of hydrocarbons. Aside from energy, manyindustries, including plastics and chemical manufacturers, rely heavilyon the availability of hydrocarbons as a feedstock for theirmanufacturing processes. Cost-effective alternatives to current sourcesof supply could help mitigate the upward pressure on energy and theseraw material costs.

PCT Pub. No. 2008/151149 describes methods and materials for cultivatingmicroalgae for the production of oil and particularly exemplifies theproduction of diesel fuel from oil produced by the microalgae Chlorellaprotothecoides. There remains a need for improved methods for producingoil in microalgae, particularly for methods that produce oils withshorter chain length and a higher degree of saturation and withoutpigments, with greater yield and efficiency. The present invention meetsthis need.

SUMMARY OF THE INVENTION

The invention provides cells of the genus Prototheca comprising anexogenous gene, and in some embodiments the cell is a strain of thespecies Prototheca moriformis, Prototheca krugani, Prototheca stagnoraor Prototheca zopfii and in other embodiment the cell has a 23S rRNAsequence with at least 70, 75, 80, 85 or 95% nucleotide identity to oneor more of SEQ ID NOs: 11-19. In some cells the exogenous gene is codingsequence and is in operable linkage with a promoter, and in someembodiments the promoter is from a gene endogenous to a species of thegenus Prototheca. In further embodiments the coding sequence encodes aprotein selected from the group consisting of a sucrose invertase, afatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, afatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehydedecarbonylase, an acyl carrier protein and a protein that impartsresistance to an antibiotic. Some embodiments of a fatty acyl-ACPthioesterase that has hydrolysis activity towards one or more fattyacyl-ACP substrates of chain length C8, C10, C12 or C14, includingacyl-ACP thioesterases with at least 50, 60, 70, 80, or 90% amino acididentity with one or more sequences selected from the group consistingof SEQ ID NOs: 59, 61, 63 and 138-140. In further embodiments the codingsequence comprises a plastid targeting sequence from microalgae, and insome embodiments the microalgae is a species of the genus Prototheca orChlorella as well as other genera from the family Chlorellaceae. In someembodiments the plastid targeting sequence has at least 20, 25, 35, 45,or 55% amino acid sequence identity to one or more of SEQ ID NOs:127-133 and is capable of targeting a protein encoded by an exogenousgene not located in the plastid genome to the plastid. In otherembodiments the promoter is upregulated in response to reduction orelimination of nitrogen in the culture media of the cell, such as atleast a 3-fold upregulation as determined by transcript abundance in acell of the genus Prototheca when the extracellular environment changesfrom containing at least 10 mM or 5 mM nitrogen to containing nonitrogen. In further embodiments the promoter comprises a segment of 50or more nucleotides of one of SEQ ID NOs: 91-102. In other embodimentsthe cell has a 23S rRNA sequence with at least 70, 75, 80, 85 or 95%nucleotide identity to one or more of SEQ ID NOs: 11-19. In otherembodiments the exogenous gene is integrated into a chromosome of thecell.

In additional embodiments of cells of the invention, the cell is of thegenus Prototheca and comprises an exogenous fatty acyl-ACP thioesterasegene and a lipid profile of at least 4% C8-C14 of total lipids of thecell, an amount of C8 that is at least 0.3% of total lipids of the cell,an amount of C10 that is at least 2% of total lipids of the cell, anamount of C12 that is at least 2% of total lipids of the cell, an amountof C14 that is at least 4% of total lipids of the cell, and an amount ofC8-C14 that is 10-30%, 20-30%, or at least 10, 20, or 30% of totallipids of the cell. In some embodiments the cell further comprises anexogenous sucrose invertase gene. In some embodiments the cell is astrain of the species Prototheca moriformis, Prototheca krugani,Prototheca stagnora or Prototheca zopfii, and in other embodiment thecell has a 23S rRNA sequence with at least 70, 75, 80, 85 or 95%nucleotide identity to one or more of SEQ ID NOs: 11-19. In otherembodiments the exogenous fatty acyl-ACP thioesterase gene is integratedinto a chromosome of the cell. Other embodiments of the inventioncomprise methods of making triglyceride compositions of a lipid profileof at least 4% C8-C14 w/w or area percent of the triglyceridecomposition, an amount of C8 that is at least 0.3% w/w or area percent,an amount of C10 that is at least 2% w/w or area percent, an amount ofC12 that is at least 2% w/w or area percent, an amount of C14 that is atleast 4% w/w or area percent, and an amount of C8-C14 that is 10-30%,20-30%, or at least 10, 20, or 30% w/w or area percent. The inventionalso comprises methods of making triglyceride compositions comprisingcultivating the foregoing cells, wherein the cells also comprise anexogenous gene encoding a sucrose invertase and sucrose is provided as acarbon source. In some embodiments the sucrose invertase has at least50, 60, 70, 80, or 90% amino acid identity to one or more of SEQ ID NOs:3, 20-29 and 90.

Embodiments of the invention include triglyceride oil compositions aswell as cells containing triglyceride oil compositions comprising alipid profile of at least 4% C8-C14 and one or more of the followingattributes: 0.1-0.4 micrograms/ml total carotenoids, less than 0.4micrograms/ml total carotenoids, less than 0.001 micrograms/ml lycopene;less than 0.02 micrograms/ml beta carotene, less than 0.02 milligrams ofchlorophyll per kilogram of oil; 0.40-0.60 milligrams of gammatocopherol per 100 grams of oil; 0.2-0.5 milligrams of totaltocotrienols per gram of oil, less than 0.4 milligrams of totaltocotrienols per gram of oil, 4-8 mg per 100 grams of oil ofcampesterol, and 40-60 mg per 100 grams of oil of stigmasterol. In someembodiments of the invention the triglyceride oil compositions have alipid profile of at least 4% C8-C14 w/w or area percent of thetriglyceride composition, an amount of C8 that is at least 0.3% w/w orarea percent, an amount of C10 that is at least 2% w/w or area percent,an amount of C12 that is at least 2% w/w or area percent, an amount ofC14 that is at least 4% w/w or area percent, and an amount of C8-C14that is 10-30%, 20-30%, or at least 10, 20, or 30% w/w or area percent.In other embodiments the triglyceride oil composition is blended with atleast one other composition selected from the group consisting of soy,rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable,Chinese tallow, olive, sunflower, cotton seed. chicken fat, beef tallow,porcine tallow, microalgae, macroalgae, Cuphea, flax, peanut, choicewhite grease, lard, Camelina sativa, mustard seed cashew nut, oats,lupine, kenaf, calendula, hemp, coffee, linseed (flax), hazelnut,euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice,tung tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba,jatropha, macadamia, Brazil nuts, avocado, petroleum, or a distillatefraction of any of the preceding oils.

Methods of the invention also include processing the aforementioned oilsof by performing one or more chemical reactions from the list consistingof transesterification, hydrogenation, hydrocracking, deoxygenation,isomerization, interesterification, hydroxylation, hydrolysis to yieldfree fatty acids, and saponification. The invention also includeshydrocarbon fuels made from hydrogenation and isomerization of theaforementioned oils and fatty acid alkyl esters made fromtransesterification of the aforementioned oils. In some embodiments thehydrocarbon fuel is made from triglyceride isolated from cells of thegenus Prototheca wherein the ASTM D86 T10-T90 distillation range is atleast 25° C. In other embodiments the fatty acid alkyl ester fuel ismade from triglyceride isolated from cells of the genus Prototheca,wherein the composition has an ASTM D6751 A1 cold soak time of less than120 seconds.

The invention also includes composition comprising (a) polysaccharidecomprising one or more monosaccharides from the list consisting of 20-30mole percent galactose; 55-65 mole percent glucose; and 5-15 molepercent mannose; (b) protein; and (c) DNA comprising a 23S rRNA sequencewith at least 70, 75, 80, 85 or 95% nucleotide identity to one or moreof SEQ ID NOs: 11-19; and (d) an exogenous gene. In some embodiments theexogenous gene is selected from a sucrose invertase and a fatty acyl-ACPthioesterase, and in further embodiments the composition furthercomprises lipid with a lipid profile of at least 4% C8-C14. In otherembodiments the composition is formulated for consumption as an animalfeed.

The invention includes recombinant nucleic acids encoding promoters thatare upregulated in response to reduction or elimination of nitrogen inthe culture media of a cell of the genus Prototheca, such as at least a3-fold upregulation as determined by transcript abundance when theextracellular environment changes from containing at least 10 mM or 5 mMnitrogen to containing no nitrogen. In some embodiments the recombinantnucleic acid comprises a segment of 50 or more nucleotides of one of SEQID NOs: 91-102. The invention also includes nucleic acid vectorscomprising an expression cassette comprising (a) a promoter that isactive in a cell of the genus Prototheca; and (b) a coding sequence inoperable linkage with the promoter wherein the coding sequence containsthe most or second most preferred codons of Table 1 for at least 20, 30,40, 50, 60, or 80% of the codons of the coding sequence. In some vectorsthe coding sequence comprises a plastid targeting sequence in-frame witha fatty acyl-ACP thioesterase, including thioesterase that havehydrolysis activity towards one or more fatty acyl-ACP substrates ofchain length C8, C10, C12 or C14. Some vectors include plastid targetingsequences that encode peptides that are capable of targeting a proteinto the plastid of a cell of the genus Prototheca, including those frommicroalgae and those wherein the plastid targeting sequence has at least20, 25, 35, 45, or 55% amino acid sequence identity to one or more ofSEQ ID NOs. 127-133 and is capable of targeting a protein to the plastidof a cell of the genus Prototheca. Additional vectors of the inventioncomprise nucleic acid sequences endogenous to the nuclear genome of acell of the genus Prototheca, wherein the sequence is at least 200nucleotides long, and some vectors comprise first and second nucleicacid sequences endogenous to the nuclear genome of a cell of the genusPrototheca, wherein the first and second sequences (a) are each at least200 nucleotides long; (b) flank the expression cassette; and (c) arelocated on the same Prototheca chromosome no more than 5, 10, 15, 20,and 50 kB apart.

The invention also includes a recombinant nucleic acid with at least 80,90, 95 or 98% nucleotide identity to one or both of SEQ ID NOs: 134-135and a recombinant nucleic acid encoding a protein with at least 80, 90,95 or 98% amino acid identity to one or both of SEQ ID NOs: 136-137.

The invention also comprises methods of producing triglyceridecompositions, comprising (a) culturing a population of cells of thegenus Prototheca in the presence of a fixed carbon source, wherein: (i)the cells contain an exogenous gene; (ii) the cells accumulate at least10, 20, 30, 40, 60, or 70% of their dry cell weight as lipid; and (iii)the fixed carbon source is selected from the group consisting of sorghumand depolymerized cellulosic material; and (b) isolating lipidcomponents from the cultured microorganisms. In some embodiments thefixed carbon source is depolymerized cellulosic material selected fromthe group consisting of corn stover, Miscanthus, forage sorghum, sugarbeet pulp and sugar cane bagasse, optionally that has been subjected towashing with water prior to the culturing step. In some methods thefixed carbon source is depolymerized cellulosic material and the glucoselevel of the depolymerized cellulosic material is concentrated to alevel of at least 300 g/liter, at least 400 g/liter, at least 500g/liter, or at least 600 g/liter of prior to the culturing step and isfed to the culture over time as the cells grow and accumulate lipid. Insome methods the exogenous gene encodes a fatty acyl-ACP thioesterasethat has hydrolysis activity towards one or more fatty acyl-ACPsubstrates of chain length C8, C10, C12 or C14, and in some methods thetriglyceride has a lipid profile of at least 4% C8-C14 and one or moreof the following attributes: 0.1-0.4 micrograms/ml total carotenoids;less than 0.02 milligrams of chlorophyll per kilogram of oil; 0.40-0.60milligrams of gamma tocopherol per 100 grams of oil; 0.2-0.5 milligramsof total tocotrienols per gram of oil, 4-8 mg per 100 grams of oil ofcampesterol, and 40-60 mg per 100 grams of oil of stigmasterol.

Further methods of the invention include producing a triglyceridecomposition, comprising: (a) culturing a population of microorganisms inthe presence of depolymerized cellulosic material, wherein: (i) thedepolymerized cellulosic material is subjected to washing with waterprior to the culturing step; (ii) the cells accumulate at least 10, 20,30, 40, 60, or 70% of their dry cell weight as lipid; and (iii) thedepolymerized cellulosic material is concentrated to at least 300, 400,500, or 600 g/liter of glucose prior to the cultivation step; (iv) themicroorganisms are cultured in a fed-batch reaction in whichdepolymerized cellulosic material of at least 300, 400, 500, or 600g/liter of glucose is fed to the microorganisms; and (b) isolating lipidcomponents from the cultured microorganisms. In some embodiments thefixed carbon source is depolymerized cellulosic material selected fromthe group consisting of corn stover, Miscanthus, forage sorghum, sugarbeet pulp and sugar cane bagasse. In further embodiments themicroorganisms are a species of the genus Prototheca and contain anexogenous gene, including a fatty acyl-ACP thioesterase that hashydrolysis activity towards one or more fatty acyl-ACP substrates ofchain length C8, C10, C12 or C14. A further method of the inventioncomprises manufacturing triglyceride oil comprising cultivating a cellthat has a 23S rRNA sequence with at least 90 or 96% nucleotide identityto SEQ ID NO: 30 in the presence of sucrose as a carbon source.

The invention also includes methods of manufacturing a chemicalcomprising performing one or more chemical reactions from the listconsisting of transesterification, hydrogenation, hydrocracking,deoxygenation, isomerization, interesterification, hydroxylation,hydrolysis, and saponification on a triglyceride oil, wherein the oilhas a lipid profile of at least 4% C8-C14 and one or more of thefollowing attributes: 0.1-0.4 micrograms/ml total carotenoids; less than0.02 milligrams of chlorophyll per kilogram of oil; 0.10-0.60 milligramsof gamma tocopherol per 100 grams of oil; 0.1-0.5 milligrams of totaltocotrienols per gram of oil, 1-8 mg per 100 grams of oil ofcampesterol, and 10-60 mg per 100 grams of oil of stigmasterol. Somemethods are performed by manufacturing the oil by cultivating a cell ofthe genus Prototheca that comprises an exogenous fatty acyl-ACPthioesterase gene that encodes a fatty acyl-ACP thioesterase havinghydrolysis activity towards one or more fatty acyl-ACP substrates ofchain length C8, C10, C12 or C14. In some methods the hydrolysisreaction is selected from the group consisting of saponification, acidhydrolysis, alkaline hydrolysis, enzymatic hydrolysis, catalytichydrolysis, and hot-compressed water hydrolysis, including a catalytichydrolysis reaction wherein the oil is split into glycerol and fattyacids. In further methods the fatty acids undergo an amination reactionto produce fatty nitrogen compounds or an ozonolysis reaction to producemono- and dibasic-acids. In some embodiments the oil undergoes atriglyceride splitting method selected from the group consisting ofenzymatic splitting and pressure splitting. In some methods acondensation reaction follows the hydrolysis reaction. Other methodsinclude performing a hydroprocessing reaction on the oil, optionallywherein the product of the hydroprocessing reaction undergoes adeoxygenation reaction or a condensation reaction prior to orsimultaneous with the hydroprocessing reaction. Some methodsadditionally include a gas removal reaction. Additional methods includeprocessing the aforementioned oils by performing a deoxygenationreaction selected from the group consisting of: a hydrogenolysisreaction, hydrogenation, a consecutive hydrogenation-hydrogenolysisreaction, a consecutive hydrogenolysis-hydrogenation reaction, and acombined hydrogenation-hydrogenolysis reaction. In some methods acondensation reaction follows the deoxygenation reaction. Other methodsinclude performing an esterification reaction on the aforementionedoils, optionally an interestification reaction or a transesterificationreaction. Other methods include performing a hydroxylation reaction onthe aforementioned oils, optionally wherein a condensation reactionfollows the hydroxylation reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate the growth curves of Prototheca species andChlorella luteoviridis strain SAG 2214 grown on sorghum as the carbonsource.

FIG. 3 shows time course growth of SAG 2214 on glucose and sucrose.

FIG. 4 shows maps of the cassettes used in Prototheca transformations,as described in Example 3.

FIG. 5 shows the results of Southern blot analysis on threetransformants of UTEX strain 1435, as described in Example 3.

FIG. 6 shows a schematic of the codon optimized and non-codon optimizedsuc2 (yeast sucrose invertase (yInv)) transgene construct. The relevantrestriction cloning sites are indicated and arrows indicate thedirection of transcription.

FIG. 7a shows the results of Prototheca moriformis grown oncellulosic-derived sugars (corn stover, beet pulp, sorghum cane,Miscanthus and glucose control). Growth is expressed in optical densitymeasurements (A750 readings).

FIG. 7b shows the results of growth experiments using Protothecamoriformis using different levels of corn stover-derived cellulosicsugar as compared to glucose/xylose control.

FIG. 7c shows the impact that xylose has on the lipid production inPrototheca cultures.

FIG. 7d shows the impact of salt concentration (Na₂SO₄) and antifoam onthe growth (in dry cell weight (DCW)) of Prototheca.

FIG. 8 shows the impact of hydrothermal treatment of various cellulosicmaterials (sugar cane bagasse, sorghum cane, Miscanthus and beet pulp)and the resulting sugar stream on the growth of Prototheca.

FIG. 9 shows decreasing levels of hydroxymethyl furfurals (HMF) andfurfurals in cellulosic biomass (sugar cane bagasse, sorghum cane,Miscanthus and beet pulp) after repeated cycles of hydrothermaltreatment.

FIG. 10 shows a schematic of a saccharification process of cellulosicmaterials to generate sugar streams suitable for use in heterotrophicoil production in a fermentor.

FIG. 11 shows decreasing levels of HMF and furfurals in exploded sugarcane bagasse after repeated cycles of hydrothermal treatment.

FIG. 12 shows a schematic of thioesterase constructs used in Protothecatransformations. The heterologous beta-tubulin (driving Neo^(R)) andglutamate dehydrogenase promoters are derived from Chlamydomonasreinhardtii and Chlorella sorokiniana, respectively. The nitratereductase 3′UTR was derived from Chlorella vulgaris. The relevantrestriction cloning sites are indicated and arrows indicate thedirection of transcription.

FIG. 13 shows a chromatogram of renewable diesel produced fromPrototheca triglyceride oil.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises from the discovery that Prototheca andcertain related microorganisms have unexpectedly advantageous propertiesfor the production of oils, fuels, and other hydrocarbon or lipidcompositions economically and in large quantities, as well as from thediscovery of methods and reagents for genetically altering thesemicroorganisms to improve these properties. The oils produced by thesemicroorganisms can be used in the transportation fuel, petrochemical,and/or food and cosmetic industries, among other applications.Transesterification of lipids yields long-chain fatty acid esters usefulas biodiesel. Other enzymatic and chemical processes can be tailored toyield fatty acids, aldehydes, alcohols, alkanes, and alkenes. In someapplications, renewable diesel, jet fuel, or other hydrocarbon compoundsare produced. The present invention also provides methods of cultivatingmicroalgae for increased productivity and increased lipid yield, and/orfor more cost-effective production of the compositions described herein.

This detailed description of the invention is divided into sections forthe convenience of the reader. Section I provides definitions of termsused herein. Section 2 provides a description of culture conditionsuseful in the methods of the invention. Section 3 provides a descriptionof genetic engineering methods and materials. Section 4 provides adescription of genetic engineering of Prototheca to enable sucroseutilization. Section 5 provides a description of genetic engineering ofPrototheca to modify lipid biosynthesis. Section 6 describes methods formaking fuels and chemicals. Section 7 discloses examples and embodimentsof the invention. The detailed description of the invention is followedby examples that illustrate the various aspects and embodiments of theinvention.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

“Active in microalgae” refers to a nucleic acid that is functional inmicroalgae. For example, a promoter that has been used to drive anantibiotic resistance gene to impart antibiotic resistance to atransgenic microalgae is active in microalgae.

“Acyl carrier protein” or “ACP” is a protein that binds a growing acylchain during fatty acid synthesis as a thiol ester at the distal thiolof the 4′-phosphopantetheine moiety and comprises a component of thefatty acid synthase complex.

“Acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acylmoiety covalently attached to coenzyme A through a thiol ester linkageat the distal thiol of the 4′-phosphopantetheine moiety of coenzyme A.

“Area Percent” refers to the area of peaks observed using FAME GC/FIDdetection methods in which every fatty acid in the sample is convertedinto a fatty acid methyl ester (FAME) prior to detection. For example, aseparate peak is observed for a fatty acid of 14 carbon atoms with nounsaturation (C14:0) compared to any other fatty acid such as C14:1. Thepeak area for each class of FAME is directly proportional to its percentcomposition in the mixture and is calculated based on the sum of allpeaks present in the sample (i.e. [area under specific peak/total areaof all measured peaks]×100). When referring to lipid profiles of oilsand cells of the invention, “at least 4% C8-C14” means that at least 4%of the total fatty acids in the cell or in the extracted glycerolipidcomposition have a chain length that includes 8, 10, 12 or 14 carbonatoms.

“Axenic” is a culture of an organism free from contamination by otherliving organisms.

“Biodiesel” is a biologically produced fatty acid alkyl ester suitablefor use as a fuel in a diesel engine.

“Biomass” is material produced by growth and/or propagation of cells.Biomass may contain cells and/or intracellular contents as well asextracellular material, includes, but is not limited to, compoundssecreted by a cell.

“Bioreactor” is an enclosure or partial enclosure in which cells arecultured, optionally in suspension.

“Catalyst” is an agent, such as a molecule or macromolecular complex,capable of facilitating or promoting a chemical reaction of a reactantto a product without becoming a part of the product. A catalystincreases the rate of a reaction, after which, the catalyst may act onanother reactant to form the product. A catalyst generally lowers theoverall activation energy required for the reaction such that itproceeds more quickly or at a lower temperature. Thus, a reactionequilibrium may be more quickly attained. Examples of catalysts includeenzymes, which are biological catalysts; heat, which is a non-biologicalcatalyst; and metals used in fossil oil refining processes.

“Cellulosic material” is the product of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar cane bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

“Co-culture”, and variants thereof such as “co-cultivate” and“co-ferment”, refer to the presence of two or more types of cells in thesame bioreactor. The two or more types of cells may both bemicroorganisms, such as microalgae, or may be a microalgal cell culturedwith a different cell type. The culture conditions may be those thatfoster growth and/or propagation of the two or more cell types or thosethat facilitate growth and/or proliferation of one, or a subset, of thetwo or more cells while maintaining cellular growth for the remainder.

“Cofactor” is any molecule, other than the substrate, required for anenzyme to carry out its enzymatic activity.

“Complementary DNA” or “cDNA” is a DNA copy of mRNA, usually obtained byreverse transcription of messenger RNA (mRNA) or amplification (e.g.,via polymerase chain reaction (“PCR”)).

“Cultivated”, and variants thereof such as “cultured” and “fermented”,refer to the intentional fostering of growth (increases in cell size,cellular contents, and/or cellular activity) and/or propagation(increases in cell numbers via mitosis) of one or more cells by use ofselected and/or controlled conditions. The combination of both growthand propagation may be termed proliferation. Examples of selected and/orcontrolled conditions include the use of a defined medium (with knowncharacteristics such as pH, ionic strength, and carbon source),specified temperature, oxygen tension, carbon dioxide levels, and growthin a bioreactor. Cultivate does not refer to the growth or propagationof microorganisms in nature or otherwise without human intervention; forexample, natural growth of an organism that ultimately becomesfossilized to produce geological crude oil is not cultivation.

“Cytolysis” is the lysis of cells in a hypotonic environment. Cytolysisis caused by excessive osmosis, or movement of water, towards the insideof a cell (hyperhydration). The cell cannot withstand the osmoticpressure of the water inside, and so it explodes.

“Delipidated meal” and “delipidated microbial biomass” is microbialbiomass after oil (including lipids) has been extracted or isolated fromit, either through the use of mechanical (i.e., exerted by an expellerpress) or solvent extraction or both. Delipidated meal has a reducedamount of oil/lipids as compared to before the extraction or isolationof oil/lipids from the microbial biomass but does contain some residualoil/lipid.

“Expression vector” or “expression construct” or “plasmid” or“recombinant DNA construct” refer to a nucleic acid that has beengenerated via human intervention, including by recombinant means ordirect chemical synthesis, with a series of specified nucleic acidelements that permit transcription and/or translation of a particularnucleic acid in a host cell. The expression vector can be part of aplasmid, virus, or nucleic acid fragment. Typically, the expressionvector includes a nucleic acid to be transcribed operably linked to apromoter.

“Exogenous gene” is a nucleic acid that codes for the expression of anRNA and/or protein that has been introduced (“transformed”) into a cell.A transformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous), relative to the cell being transformed.Thus, an exogenous gene can include a homologous gene that occupies adifferent location in the genome of the cell or is under differentcontrol, relative to the endogenous copy of the gene. An exogenous genemay be present in more than one copy in the cell. An exogenous gene maybe maintained in a cell as an insertion into the genome or as anepisomal molecule.

“Exogenously provided” refers to a molecule provided to the culturemedia of a cell culture.

“Expeller pressing” is a mechanical method for extracting oil from rawmaterials such as soybeans and rapeseed. An expeller press is a screwtype machine, which presses material through a caged barrel-like cavity.Raw materials enter one side of the press and spent cake exits the otherside while oil seeps out between the bars in the cage and is collected.The machine uses friction and continuous pressure from the screw drivesto move and compress the raw material. The oil seeps through smallopenings that do not allow solids to pass through. As the raw materialis pressed, friction typically causes it to heat up.

“Fatty acyl-ACP thioesterase” is an enzyme that catalyzes the cleavageof a fatty acid from an acyl carrier protein (ACP) during lipidsynthesis.

“Fatty acyl-CoA/aldehyde reductase” is an enzyme that catalyzes thereduction of an acyl-CoA molecule to a primary alcohol.

“Fatty acyl-CoA reductase” is an enzyme that catalyzes the reduction ofan acyl-CoA molecule to an aldehyde.

“Fatty aldehyde decarbonylase” is an enzyme that catalyzes theconversion of a fatty aldehyde to an alkane.

“Fatty aldehyde reductase” is an enzyme that catalyzes the reduction ofan aldehyde to a primary alcohol.

“Fixed carbon source” is a molecule(s) containing carbon, typically anorganic molecule, that is present at ambient temperature and pressure insolid or liquid form in a culture media that can be utilized by amicroorganism cultured therein.

“Homogenate” is biomass that has been physically disrupted.

“Hydrocarbon” is (a) a molecule containing only hydrogen and carbonatoms wherein the carbon atoms are covalently linked to form a linear,branched, cyclic, or partially cyclic backbone to which the hydrogenatoms are attached. The molecular structure of hydrocarbon compoundsvaries from the simplest, in the form of methane (CH₄), which is aconstituent of natural gas, to the very heavy and very complex, such assome molecules such as asphaltenes found in crude oil, petroleum, andbitumens. Hydrocarbons may be in gaseous, liquid, or solid form, or anycombination of these forms, and may have one or more double or triplebonds between adjacent carbon atoms in the backbone. Accordingly, theterm includes linear, branched, cyclic, or partially cyclic alkanes,alkenes, lipids, and paraffin. Examples include propane, butane,pentane, hexane, octane, and squalene.

“Hydrogen:carbon ratio” is the ratio of hydrogen atoms to carbon atomsin a molecule on an atom-to-atom basis. The ratio may be used to referto the number of carbon and hydrogen atoms in a hydrocarbon molecule.For example, the hydrocarbon with the highest ratio is methane CH₄(4:1).

“Hydrophobic fraction” is the portion, or fraction, of a material thatis more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

“Increase lipid yield” refers to an increase in the productivity of amicrobial culture by, for example, increasing dry weight of cells perliter of culture, increasing the percentage of cells that constitutelipid, or increasing the overall amount of lipid per liter of culturevolume per unit time.

“Inducible promoter” is a promoter that mediates transcription of anoperably linked gene in response to a particular stimulus.

“In operable linkage” is a functional linkage between two nucleic acidsequences, such a control sequence (typically a promoter) and the linkedsequence (typically a sequence that encodes a protein, also called acoding sequence). A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

“In situ” means “in place” or “in its original position”.

“Limiting concentration of a nutrient” is a concentration of a compoundin a culture that limits the propagation of a cultured organism. A“non-limiting concentration of a nutrient” is a concentration thatsupports maximal propagation during a given culture period. Thus, thenumber of cells produced during a given culture period is lower in thepresence of a limiting concentration of a nutrient than when thenutrient is non-limiting. A nutrient is said to be “in excess” in aculture, when the nutrient is present at a concentration greater thanthat which supports maximal propagation.

“Lipase” is a water-soluble enzyme that catalyzes the hydrolysis ofester bonds in water-insoluble, lipid substrates. Lipases catalyze thehydrolysis of lipids into glycerols and fatty acids.

“Lipid modification enzyme” refers to an enzyme that alters the covalentstructure of a lipid. Examples of lipid modification enzymes include alipase, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehydereductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, and afatty aldehyde decarbonylase.

“Lipid pathway enzyme” is any enzyme that plays a role in lipidmetabolism, i.e., either lipid synthesis, modification, or degradation,and any proteins that chemically modify lipids, as well as carrierproteins.

“Lipids” are a class of molecules that are soluble in nonpolar solvents(such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties, because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” are a subgroup of lipids called “triacylglycerides.”

“Lysate” is a solution containing the contents of lysed cells.

“Lysis” is the breakage of the plasma membrane and optionally the cellwall of a biological organism sufficient to release at least someintracellular content, often by mechanical, viral or osmotic mechanismsthat compromise its integrity.

“Lysing” is disrupting the cellular membrane and optionally the cellwall of a biological organism or cell sufficient to release at leastsome intracellular content.

“Microalgae” is a eukarytotic microbial organism that contains achloroplast or plastid, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae include unicellular organisms that separate from sister cellsshortly after cell division, such as Chlamydomonas, as well as microbessuch as, for example, Volvox, which is a simple multicellularphotosynthetic microbe of two distinct cell types. Microalgae includecells such as Chlorella, Dunaliella, and Prototheca. Microalgae alsoinclude other microbial photosynthetic organisms that exhibit cell-celladhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae alsoinclude obligate heterotrophic microorganisms that have lost the abilityto perform photosynthesis, such as certain dinoflagellate algae speciesand species of the genus Prototheca.

“Microorganism” and “microbe” are microscopic unicellular organisms.

“Naturally co-expressed” with reference to two proteins or genes meansthat the proteins or their genes are co-expressed naturally in a tissueor organism from which they are derived, e.g., because the genesencoding the two proteins are under the control of a common regulatorysequence or because they are expressed in response to the same stimulus.

“Osmotic shock” is the rupture of cells in a solution following a suddenreduction in osmotic pressure. Osmotic shock is sometimes induced torelease cellular components of such cells into a solution.

“Polysaccharide-degrading enzyme” is any enzyme capable of catalyzingthe hydrolysis, or saccharification, of any polysaccharide. For example,cellulases catalyze the hydrolysis of cellulose.

“Polysaccharides” or “glycans” are carbohydrates made up ofmonosaccharides joined together by glycosidic linkages. Cellulose is apolysaccharide that makes up certain plant cell walls. Cellulose can bedepolymerized by enzymes to yield monosaccharides such as xylose andglucose, as well as larger disaccharides and oligosaccharides.

“Promoter” is a nucleic acid control sequence that directs transcriptionof a nucleic acid. As used herein, a promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements, which can belocated as much as several thousand base pairs from the start site oftranscription.

“Recombinant” is a cell, nucleic acid, protein or vector, that has beenmodified due to the introduction of an exogenous nucleic acid or thealteration of a native nucleic acid. Thus, e.g., recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes differently than those genesare expressed by a non-recombinant cell. A “recombinant nucleic acid” isa nucleic acid originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,or otherwise is in a form not normally found in nature. Recombinantnucleic acids may be produced, for example, to place two or more nucleicacids in operable linkage. Thus, an isolated nucleic acid or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined in nature, are both considered recombinant for thepurposes of this invention. Once a recombinant nucleic acid is made andintroduced into a host cell or organism, it may replicate using the invivo cellular machinery of the host cell; however, such nucleic acids,once produced recombinantly, although subsequently replicatedintracellularly, are still considered recombinant for purposes of thisinvention. Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid.

“Renewable diesel” is a mixture of alkanes (such as C10:0, C12:0, C14:0,C16:0 and C18:0) produced through hydrogenation and deoxygenation oflipids.

“Saccharification” is a process of converting biomass, usuallycellulosic or lignocellulosic biomass, into monomeric sugars, such asglucose and xylose. “Saccharified” or “depolymerized” cellulosicmaterial or biomass refers to cellulosic material or biomass that hasbeen converted into monomeric sugars through saccharification.

“Sonication” is a process of disrupting biological materials, such as acell, by use of sound wave energy.

“Species of furfural” is 2-furancarboxaldehyde or a derivative thatretains the same basic structural characteristics.

“Stover” is the dried stalks and leaves of a crop remaining after agrain has been harvested.

“Sucrose utilization gene” is a gene that, when expressed, aids theability of a cell to utilize sucrose as an energy source. Proteinsencoded by a sucrose utilization gene are referred to herein as “sucroseutilization enzymes” and include sucrose transporters, sucroseinvertases, and hexokinases such as glucokinases and fructokinases.

II. CULTIVATION

The present invention generally relates to cultivation of Protothecastrains, particularly recombinant Prototheca strains, for the productionof lipid. For the convenience of the reader, this section is subdividedinto subsections. Subsection 1 describes Prototheca species and strainsand how to identify new Prototheca species and strains and relatedmicroalgae by genomic DNA comparison. Subsection 2 describes bioreactorsuseful for cultivation. Subsection 3 describes media for cultivation.Subsection 4 describes oil production in accordance with illustrativecultivation methods of the invention.

1. Prototheca Species and Strains

Prototheca is a remarkable microorganism for use in the production oflipid, because it can produce high levels of lipid, particularly lipidsuitable for fuel production. The lipid produced by Prototheca hashydrocarbon chains of shorter chain length and a higher degree ofsaturation than that produced by other microalgae. Moreover, Protothecalipid is generally free of pigment (low to undetectable levels ofchlorophyll and certain carotenoids) and in any event contains much lesspigment than lipid from other microalgae. Moreover, recombinantPrototheca cells provided by the invention can be used to produce lipidin greater yield and efficiency, and with reduced cost, relative to theproduction of lipid from other microorganisms. Illustrative Protothecastrains for use in the methods of the invention include In addition,this microalgae grows heterotrophically and can be geneticallyengineered as Prototheca wickerhamii, Prototheca stagnora (includingUTEX 327), Prototheca portoricensis, Prototheca moriformis (includingUTEX strains 1441, 1435), and Prototheca zopfii. Species of the genusPrototheca are obligate heterotrophs.

Species of Prototheca for use in the invention can be identified byamplification of certain target regions of the genome. For example,identification of a specific Prototheca species or strain can beachieved through amplification and sequencing of nuclear and/orchloroplast DNA using primers and methodology using any region of thegenome, for example using the methods described in Wu et al., Bot. Bull.Acad. Sin. (2001) 42:115-121 Identification of Chlorella spp. isolatesusing ribosomal DNA sequences. Well established methods of phylogeneticanalysis, such as amplification and sequencing of ribosomal internaltranscribed spacer (ITS1 and ITS2 rDNA), 23S rRNA, 18S rRNA, and otherconserved genomic regions can be used by those skilled in the art toidentify species of not only Prototheca, but other hydrocarbon and lipidproducing organisms with similar lipid profiles and productioncapability. For examples of methods of identification and classificationof algae also see for example Genetics, 2005 August; 170(4):1601-10 andRNA, 2005 April; 11(4):361-4.

Thus, genomic DNA comparison can be used to identify suitable species ofmicroalgae to be used in the present invention. Regions of conservedgenomic DNA, such as but not limited to DNA encoding for 23S rRNA, canbe amplified from microalgal species and compared to consensus sequencesin order to screen for microalgal species that are taxonomically relatedto the preferred microalgae used in the present invention. Examples ofsuch DNA sequence comparison for species within the Prototheca genus areshown below. Genomic DNA comparison can also be useful to identifymicroalgal species that have been misidentified in a strain collection.Often a strain collection will identify species of microalgae based onphenotypic and morphological characteristics. The use of thesecharacteristics may lead to miscategorization of the species or thegenus of a microalgae. The use of genomic DNA comparison can be a bettermethod of categorizing microalgae species based on their phylogeneticrelationship.

Microalgae for use in the present invention typically have genomic DNAsequences encoding for 23S rRNA that have at least 99%, least 95%, atleast 90%, or at least 85% nucleotide identity to at least one of thesequences listed in SEQ ID NOs: 11-19.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Other considerations affecting the selection of microorganisms for usein the invention include, in addition to production of suitable lipidsor hydrocarbons for production of oils, fuels, and oleochemicals: (1)high lipid content as a percentage of cell weight; (2) ease of growth;(3) ease of genetic engineering; and (4) ease of biomass processing. Inparticular embodiments, the wild-type or genetically engineeredmicroorganism yields cells that are at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, or at least 70% or morelipid. Preferred organisms grow heterotrophically (on sugars in theabsence of light).

2. Bioreactor

Microorganisms are cultured both for purposes of conducting geneticmanipulations and for production of hydrocarbons (e.g., lipids, fattyacids, aldehydes, alcohols, and alkanes). The former type of culture isconducted on a small scale and initially, at least, under conditions inwhich the starting microorganism can grow. Culture for purposes ofhydrocarbon production is usually conducted on a large scale (e.g.,10,000 L, 40,000 L, 100,000 L or larger bioreactors) in a bioreactor.Prototheca are typically cultured in the methods of the invention inliquid media within a bioreactor. Typically, the bioreactor does notallow light to enter.

The bioreactor or fermentor is used to culture microalgal cells throughthe various phases of their physiological cycle. Bioreactors offer manyadvantages for use in heterotrophic growth and propagation methods. Toproduce biomass for use in food, microalgae are preferably fermented inlarge quantities in liquid, such as in suspension cultures as anexample. Bioreactors such as steel fermentors can accommodate very largeculture volumes (40,000 liter and greater capacity bioreactors are usedin various embodiments of the invention). Bioreactors also typicallyallow for the control of culture conditions such as temperature, pH,oxygen tension, and carbon dioxide levels. For example, bioreactors aretypically configurable, for example, using ports attached to tubing, toallow gaseous components, like oxygen or nitrogen, to be bubbled througha liquid culture. Other culture parameters, such as the pH of theculture media, the identity and concentration of trace elements, andother media constituents can also be more readily manipulated using abioreactor.

Bioreactors can be configured to flow culture media though thebioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments, for example,media can be infused into the bioreactor after inoculation but beforethe cells reach a desired density. In other instances, a bioreactor isfilled with culture media at the beginning of a culture, and no moreculture media is infused after the culture is inoculated. In otherwords, the microalgal biomass is cultured in an aqueous medium for aperiod of time during which the microalgae reproduce and increase innumber; however, quantities of aqueous culture medium are not flowedthrough the bioreactor throughout the time period. Thus in someembodiments, aqueous culture medium is not flowed through the bioreactorafter inoculation.

Bioreactors equipped with devices such as spinning blades and impellers,rocking mechanisms, stir bars, means for pressurized gas infusion can beused to subject microalgal cultures to mixing. Mixing may be continuousor intermittent. For example, in some embodiments, a turbulent flowregime of gas entry and media entry is not maintained for reproductionof microalgae until a desired increase in number of said microalgae hasbeen achieved.

Bioreactor ports can be used to introduce, or extract, gases, solids,semisolids, and liquids, into the bioreactor chamber containing themicroalgae. While many bioreactors have more than one port (for example,one for media entry, and another for sampling), it is not necessary thatonly one substance enter or leave a port. For example, a port can beused to flow culture media into the bioreactor and later used forsampling, gas entry, gas exit, or other purposes. Preferably, a samplingport can be used repeatedly without altering compromising the axenicnature of the culture. A sampling port can be configured with a valve orother device that allows the flow of sample to be stopped and started orto provide a means of continuous sampling. Bioreactors typically have atleast one port that allows inoculation of a culture, and such a port canalso be used for other purposes such as media or gas entry.

Bioreactors ports allow the gas content of the culture of microalgae tobe manipulated. To illustrate, part of the volume of a bioreactor can begas rather than liquid, and the gas inlets of the bioreactor to allowpumping of gases into the bioreactor. Gases that can be beneficiallypumped into a bioreactor include air, air/CO₂ mixtures, noble gases,such as argon, and other gases. Bioreactors are typically equipped toenable the user to control the rate of entry of a gas into thebioreactor. As noted above, increasing gas flow into a bioreactor can beused to increase mixing of the culture.

Increased gas flow affects the turbidity of the culture as well.Turbulence can be achieved by placing a gas entry port below the levelof the aqueous culture media so that gas entering the bioreactor bubblesto the surface of the culture. One or more gas exit ports allow gas toescape, thereby preventing pressure buildup in the bioreactor.Preferably a gas exit port leads to a “one-way” valve that preventscontaminating microorganisms from entering the bioreactor.

3. Media

Microalgal culture media typically contains components such as a fixednitrogen source, a fixed carbon source, trace elements, optionally abuffer for pH maintenance, and phosphate (typically provided as aphosphate salt). Other components can include salts such as sodiumchloride, particularly for seawater microalgae. Nitrogen sources includeorganic and inorganic nitrogen sources, including, for example, withoutlimitation, molecular nitrogen, nitrate, nitrate salts, ammonia (pure orin salt form, such as, (NH₄)₂SO₄ and NH₄OH), protein, soybean meal,cornsteep liquor, and yeast extract. Examples of trace elements includezinc, boron, cobalt, copper, manganese, and molybdenum in, for example,the respective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂Oand (NH₄)₆Mo₇O₂₄.4H₂O.

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentscan be difficult to predict. In some cases, certain strains ofmicroorganisms may be unable to grow on a particular growth mediumbecause of the presence of some inhibitory component or the absence ofsome essential nutritional requirement required by the particular strainof microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin, 1 University StationA6700, Austin, Tex., 78712-0183, for its culture collection of algae(UTEX). For example, various fresh water and salt water media includethose described in PCT Pub. No. 2008/151149, incorporated herein byreference.

In a particular example, Proteose Medium is suitable for axeniccultures, and a 1 L volume of the medium (pH ˜6.8) can be prepared byaddition of 1 g of proteose peptone to 1 liter of Bristol Medium.Bristol medium comprises 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3 mMMgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaCl in an aqueoussolution. For 1.5% agar medium, 15 g of agar can be added to 1 L of thesolution. The solution is covered and autoclaved, and then stored at arefrigerated temperature prior to use. Another example is the Protothecaisolation medium (PIM), which comprises 10 g/L postassium hydrogenphthalate (KHP), 0.9 g/L sodium hydroxide, 0.1 g/L magnesium sulfate,0.2 g/L potassium hydrogen phosphate, 0.3 g/L ammonium chloride, 10 g/Lglucose 0.001 g/L thiamine hydrochloride, 20 g/L agar, 0.25 g/L5-fluorocytosine, at a pH in the range of 5.0 to 5.2 (see Pore, 1973,App. Microbiology, 26: 648-649). Other suitable media for use with themethods of the invention can be readily identified by consulting the URLidentified above, or by consulting other organizations that maintaincultures of microorganisms, such as SAG, CCAP, or CCALA. SAG refers tothe Culture Collection of Algae at the University of Gottingen(Gottingen, Germany), CCAP refers to the culture collection of algae andprotozoa managed by the Scottish Association for Marine Science(Scotland, United Kingdom), and CCALA refers to the culture collectionof algal laboratory at the Institute of Botany (T{hacek over(r)}ebo{hacek over (n)}, Czech Republic). Additionally, U.S. Pat. No.5,900,370 describes media formulations and conditions suitable forheterotrophic fermentation of Prototheca species.

For oil production, selection of a fixed carbon source is important, asthe cost of the fixed carbon source must be sufficiently low to make oilproduction economical. Thus, while suitable carbon sources include, forexample, acetate, floridoside, fructose, galactose, glucuronic acid,glucose, glycerol, lactose, mannose, N-acetylglucosamine, rhamnose,sucrose, and/or xylose, selection of feedstocks containing thosecompounds is an important aspect of the methods of the invention.Suitable feedstocks useful in accordance with the methods of theinvention include, for example, black liquor, corn starch, depolymerizedcellulosic material, milk whey, molasses, potato, sorghum, sucrose,sugar beet, sugar cane, rice, and wheat. Carbon sources can also beprovided as a mixture, such as a mixture of sucrose and depolymerizedsugar beet pulp. The one or more carbon source(s) can be supplied at aconcentration of at least about 50 μM, at least about 100 μM, at leastabout 500 μM, at least about 5 mM, at least about 50 mM, and at leastabout 500 mM, of one or more exogenously provided fixed carbonsource(s). Carbon sources of particular interest for purposes of thepresent invention include cellulose (in a depolymerized form), glycerol,sucrose, and sorghum, each of which is discussed in more detail below.

In accordance with the present invention, microorganisms can be culturedusing depolymerized cellulosic biomass as a feedstock. Cellulosicbiomass (e.g., stover, such as corn stover) is inexpensive and readilyavailable; however, attempts to use this material as a feedstock foryeast have failed. In particular, such feedstocks have been found to beinhibitory to yeast growth, and yeast cannot use the 5-carbon sugarsproduced from cellulosic materials (e.g., xylose from hemi-cellulose).By contrast, microalgae can grow on processed cellulosic material.Cellulosic materials generally include about 40-60% cellulose; about20-40% hemicellulose; and 10-30% lignin.

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

Cellulosic materials are treated to increase the efficiency with whichthe microbe can utilize the sugar(s) contained within the materials. Theinvention provides novel methods for the treatment of cellulosicmaterials after acid explosion so that the materials are suitable foruse in a heterotrophic culture of microbes (e.g., microalgae andoleaginous yeast). As discussed above, lignocellulosic biomass iscomprised of various fractions, including cellulose, a crystallinepolymer of beta 1,4 linked glucose (a six-carbon sugar), hemicellulose,a more loosely associated polymer predominantly comprised of xylose (afive-carbon sugar) and to a lesser extent mannose, galactose, arabinose,lignin, a complex aromatic polymer comprised of sinapyl alcohol and itsderivatives, and pectins, which are linear chains of an alpha 1,4 linkedpolygalacturonic acid. Because of the polymeric structure of celluloseand hemicellulose, the sugars (e.g., monomeric glucose and xylose) inthem are not in a form that can be efficiently used (metabolized) bymany microbes. For such microbes, further processing of the cellulosicbiomass to generate the monomeric sugars that make up the polymers canbe very helpful to ensuring that the cellulosic materials areefficiently utilized as a feedstock (carbon source).

Celluose or cellulosic biomass is subjected to a process, termed“explosion”, in which the biomass is treated with dilute sulfuric (orother) acid at elevated temperature and pressure. This processconditions the biomass such that it can be efficiently subjected toenzymatic hydrolysis of the cellulosic and hemicellulosic fractions intoglucose and xylose monomers. The resulting monomeric sugars are termedcellulosic sugars. Cellulosic sugars can subsequently be utilized bymicroorganisms to produce a variety of metabolites (e.g., lipid). Theacid explosion step results in a partial hydrolysis of the hemicellulosefraction to constitutent monosaccharides. These sugars can be completelyliberated from the biomass with further treatment. In some embodiments,the further treatment is a hydrothermal treatment that includes washingthe exploded material with hot water, which removes contaminants such assalts. This step is not necessary for cellulosic ethanol fermentationsdue to the more dilute sugar concentrations used in such processes. Inother embodiments, the further treatment is additional acid treatment.In still other embodiments, the further treatment is enzymatichydrolysis of the exploded material. These treatments can also be usedin any combination. The type of treatment can affect the type of sugarsliberated (e.g., five carbon sugars versus six carbon sugars) and thestage at which they are liberated in the process. As a consequence,different streams of sugars, whether they are predominantly five-carbonor six-carbon, can be created. These enriched five-carbon or six-carbonstreams can thus be directed to specific microorganisms with differentcarbon utilization cabilities.

The methods of the present invention typically involve fermentation tohigher cell densities than what is achieved in ethanol fermentation.Because of the higher densities of the cultures for heterotrophiccellulosic oil production, the fixed carbon source (e.g., the cellulosicderived sugar stream(s)) is preferably in a concentrated form. Theglucose level of the depolymerized cellulosic material is preferably atleast 300 g/liter, at least 400 g/liter, at least 500 g/liter or atleast 600 g/liter prior to the cultivation step, which is optionally afed batch cultivation in which the material is fed to the cells overtime as the cells grow and accumulate lipid. Cellulosic sugar streamsare not used at or near this concentration range in the production ofcellulosic ethanol. Thus, in order to generate and sustain the very highcell densities during the production of lignocellulosic oil, the carbonfeedstock(s) must be delivered into the heterotrophic cultures in ahighly concentrated form. However, any component in the feedstream thatis not a substrate for, and is not metabolized by, the oleaginousmicroorganism will accumulate in the bioreactor, which can lead toproblems if the component is toxic or inhibitory to production of thedesired end product. While ligin and lignin-derived by-products,carbohydrate-derived byproducts such as furfurals and hydroxymethylfurfurals and salts derived from the generation of the cellulosicmaterials (both in the explosion process and the subsequentneutralization process), and even non-metabolized pentose/hexose sugarscan present problems in ethanolic fermentations, these effects areamplified significantly in a process in which their concentration in theinitial feedstock is high. To achieve sugar concentrations in the 300g/L range (or higher) for six-carbon sugars that may be used in largescale production of lignocellulosic oil described in the presentinvention, the concentration of these toxic materials can be 20 timeshigher than the concentrations typically present in ethanolicfermentations of cellulosic biomass.

The explosion process treatment of the cellulosic material utilizessignificant amounts of sulfuric acid, heat and pressure, therebyliberating by-products of carbohydrates, namely furfurals andhydroxymethyl furfurals. Furfurals and hydroxymethyl furfurals areproduced during hydrolysis of hemicellulose through dehydration ofxylose into furfural and water. In some embodiments of the presentinvention, these by-products (e.g., furfurals and hydroxymethylfurfurals) are removed from the saccharified lignocellulosic materialprior to introduction into the bioreactor. In certain embodiments of thepresent invention, the process for removal of the by-products ofcarbohydrates is hydrothermal treatment of the exploded cellulosicmaterials. In addition, the present invention provides methods in whichstrains capable of tolerating compounds such as furfurals orhydroxymethyl furfurals are used for lignocellulosic oil production. Inanother embodiment, the present invention also provides methods andmicroorganisms that are not only capable of tolerating furfurals in thefermentation media, but are actually able to metabolize theseby-products during the production of lignocellulosic oil.

The explosion process also generates significant levels of salts. Forexample, typical conditions for explosion can result in conductivites inexcess of 5 mS/cm when the exploded cellulosic biomass is resuspended ata ratio of 10:1 water:solids (dry weight). In certain embodiments of thepresent invention, the diluted exploded biomass is subjected toenzymatic saccharification, and the resulting supernatant isconcentrated up to 25 fold for use in the bioreactor. The salt level (asmeasured by conductivity) in the concentrated sugar stream(s) can beunacceptably high (up to 1.5 M Na⁺ equivalents). Additional salts aregenerated upon neutralization of the exploded materials for thesubsequent enzymatic saccharification process as well. The presentinvention provides methods for removing these salts so that theresulting concentrated cellulosic sugar stream(s) can be used inheterotrophic processes for producing lignocellulosic oil. In someembodiments, the method of removing these salts is deionization withresins, such as, but not limited to, DOWEX Marathon MR3. In certainembodiments, the deionization with resin step occurs before sugarconcentration or pH adjustment and hydrothermal treatment of biomassprior to saccharification, or any combination of the preceding; in otherembodiments, the step is conducted after one or more of these processes.In other embodiments, the explosion process itself is changed so as toavoid the generation of salts at unacceptably high levels. For example,a suitable alternative to sulfuric acid (or other acid) explosion of thecellulosic biomass is mechanical pulping to render the cellulosicbiomass receptive to enzymatic hydrolysis (saccharification). In stillother embodiments, native strains of microorganisms resistant to highlevels of salts or genetically engineered strains with resistance tohigh levels of salts are used.

A preferred embodiment for the process of preparing of explodedcellulosic biomass for use in heterotrophic lignocellulosic oilproduction using oleaginous microbes is diagramed in FIG. 10. Step I.comprises adjusting the pH of the resuspended exploded cellulosicbiomass to the range of 5.0-5.3 followed by washing the cellulosicbiomass three times. This washing step can be accomplished by a varietyof means including the use of desalting and ion exchange resins, reverseomosis, hydrothermal treatment (as described above), or just repeatedre-suspension and centrifugation in deionized water. This wash stepresults in a cellulosic stream whose conductivity is between 100-300μS/cm and the removal of significant amounts of furfurals andhydroxymethyl furfurals. Decants from this wash step can be saved toconcentrate five-carbon sugars liberated from the hemicellulosefraction. Step II comprises enzymatic saccharification of the washedcellulosic biomass. In a preferred embodiment, Accellerase (Genencor) isused. Step III comprises the recovery of sugars via centrifugation ordecanting and rinsing of the saccharified biomass. The resulting biomass(solids) is an energy dense, lignin rich component that can be used asfuel or sent to waste. The recovered sugar stream in thecentrifugation/decanting and rinse process is collected. Step IVcomprises microfiltration to remove contaminating solids with recoveryof the permeate. Step V comprises a concentration step which can beaccomplished using a vacuum evaporator. This step can optionally includethe addition of antifoam agents such as P′2000 (Sigma/Fluka), which issometimes necessary due to the protein content of the resulting sugarfeedstock.

In another embodiment of the methods of the invention, the carbon sourceis glycerol, including acidulated and non-acidulated glycerol byproductfrom biodiesel transesterification. In one embodiment, the carbon sourceincludes glycerol and at least one other carbone source. In some cases,all of the glycerol and the at least one other fixed carbon source areprovided to the microorganism at the beginning of the fermentation. Insome cases, the glycerol and the at least one other fixed carbon sourceare provided to the microorganism simultaneously at a predeterminedratio. In some cases, the glycerol and the at least one other fixedcarbon source are fed to the microbes at a predetermined rate over thecourse of fermentation.

Some microalgae undergo cell division faster in the presence of glycerolthan in the presence of glucose (see PCT Pub. No. 2008/151149). In theseinstances, two-stage growth processes in which cells are first fedglycerol to rapidly increase cell density, and are then fed glucose toaccumulate lipids can improve the efficiency with which lipids areproduced. The use of the glycerol byproduct of the transesterificationprocess provides significant economic advantages when put back into theproduction process. Other feeding methods are provided as well, such asmixtures of glycerol and glucose. Feeding such mixtures also capturesthe same economic benefits. In addition, the invention provides methodsof feeding alternative sugars to microalgae such as sucrose in variouscombinations with glycerol.

In another embodiment of the methods of the invention, the carbon sourceis sucrose, including a complex feedstock containing sucrose, such asthick cane juice from sugar cane processing. In one embodiment, theculture medium further includes at least one sucrose utilization enzyme.In some cases, the culture medium includes a sucrose invertase. In oneembodiment, the sucrose invertase enzyme is a secrectable sucroseinvertase enzyme encoded by an exogenous sucrose invertase geneexpressed by the population of microorganisms. Thus, in some cases, asdescribed in more detail in Section IV, below, the microalgae has beengenetically engineered to express a sucrose utilization enzyme, such asa sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase,or a fructokinase.

Complex feedstocks containing sucrose include waste molasses from sugarcane processing; the use of this low-value waste product of sugar caneprocessing can provide significant cost savings in the production ofhydrocarbons and other oils. Another complex feedstock containingsucrose that is useful in the methods of the invention is sorghum,including sorghum syrup and pure sorghum. Sorghum syrup is produced fromthe juice of sweet sorghum cane. Its sugar profile consists of mainlyglucose (dextrose), fructose and sucrose.

4. Oil Production

For the production of oil in accordance with the methods of theinvention, it is preferable to culture cells in the dark, as is thecase, for example, when using extremely large (40,000 liter and higher)fermentors that do not allow light to strike the culture. Protothecaspecies are grown and propagated for the production of oil in a mediumcontaining a fixed carbon source and in the absence of light; suchgrowth is known as heterotrophic growth.

As an example, an inoculum of lipid-producing microalgal cells areintroduced into the medium; there is a lag period (lag phase) before thecells begin to propagate. Following the lag period, the propagation rateincreases steadily and enters the log, or exponential, phase. Theexponential phase is in turn followed by a slowing of propagation due todecreases in nutrients such as nitrogen, increases in toxic substances,and quorum sensing mechanisms. After this slowing, propagation stops,and the cells enter a stationary phase or steady growth state, dependingon the particular environment provided to the cells. For obtaining lipidrich biomass, the culture is typically harvested well after then end ofthe exponential phase, which may be terminated early by allowingnitrogen or another key nutrient (other than carbon) to become depleted,forcing the cells to convert the carbon sources, present in excess, tolipid. Culture condition parameters can be manipulated to optimize totaloil production, the combination of lipid species produced, and/orproduction of a specific oil.

As discussed above, a bioreactor or fermentor is used to allow cells toundergo the various phases of their growth cycle. As an example, aninoculum of lipid-producing cells can be introduced into a mediumfollowed by a lag period (lag phase) before the cells begin growth.Following the lag period, the growth rate increases steadily and entersthe log, or exponential, phase. The exponential phase is in turnfollowed by a slowing of growth due to decreases in nutrients and/orincreases in toxic substances. After this slowing, growth stops, and thecells enter a stationary phase or steady state, depending on theparticular environment provided to the cells. Lipid production by cellsdisclosed herein can occur during the log phase or thereafter, includingthe stationary phase wherein nutrients are supplied, or still available,to allow the continuation of lipid production in the absence of celldivision.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 20% by weight of lipid,preferably at least about 40% by weight, more preferably at least about50% by weight, and most preferably at least about 60% by weight. Processconditions can be adjusted to increase the yield of lipids suitable fora particular use and/or to reduce production cost. For example, incertain embodiments, a microalgae is cultured in the presence of alimiting concentration of one or more nutrients, such as, for example,nitrogen, phosphorous, or sulfur, while providing an excess of fixedcarbon energy such as glucose. Nitrogen limitation tends to increasemicrobial lipid yield over microbial lipid yield in a culture in whichnitrogen is provided in excess. In particular embodiments, the increasein lipid yield is at least about: 10%, 50%, 100%, 200%, or 500%. Themicrobe can be cultured in the presence of a limiting amount of anutrient for a portion of the total culture period or for the entireperiod. In particular embodiments, the nutrient concentration is cycledbetween a limiting concentration and a non-limiting concentration atleast twice during the total culture period. Lipid content of cells canbe increased by continuing the culture for increased periods of timewhile providing an excess of carbon, but limiting or no nitrogen.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example:biotin, pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingcontructs and techniques such as those described above.

The specific examples of bioreactors, culture conditions, andheterotrophic growth and propagation methods described herein can becombined in any suitable manner to improve efficiencies of microbialgrowth and lipid and/or protein production.

Microalgal biomass with a high percentage of oil/lipid accumulation bydry weight has been generated using different methods of culture, whichare known in the art (see PCT Pub. No. 2008/151149). Microalgal biomassgenerated by the culture methods described herein and useful inaccordance with the present invention comprises at least 10% microalgaloil by dry weight. In some embodiments, the microalgal biomass comprisesat least 25%, at least 50%, at least 55%, or at least 60% microalgal oilby dry weight. In some embodiments, the microalgal biomass contains from10-90% microalgal oil, from 25-75% microalgal oil, from 40-75%microalgal oil, or from 50-70% microalgal oil by dry weight.

The microalgal oil of the biomass described herein, or extracted fromthe biomass for use in the methods and compositions of the presentinvention can comprise glycerolipids with one or more distinct fattyacid ester side chains. Glycerolipids are comprised of a glycerolmolecule esterified to one, two or three fatty acid molecules, which canbe of varying lengths and have varying degrees of saturation. The lengthand saturation characteristics of the fatty acid molecules (and themicroalgal oils) can be manipulated to modify the properties orproportions of the fatty acid molecules in the microalgal oils of thepresent invention via culture conditions or via lipid pathwayengineering, as described in more detail in Section IV, below. Thus,specific blends of algal oil can be prepared either within a singlespecies of algae by mixing together the biomass or algal oil from two ormore species of microalgae, or by blending algal oil of the inventionwith oils from other sources such as soy, rapeseed, canola, palm, palmkernel, coconut, corn, waste vegetable, Chinese tallow, olive,sunflower, cottonseed, chicken fat, beef tallow, porcine tallow,microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice whitegrease, lard, Camelina sativa, mustard seed, cashew nut, oats, lupine,kenaf, calendula, help, coffee, linseed (flax), hazelnut, euphorbia,pumpkin seed, coriander, camellia, sesame, safflower, rice, tung tree,cocoa, copra, pium poppy, castor beans, pecan, jojoba, macadamia, Brazilnuts, avocado, petroleum, or a distillate fraction of any of thepreceding oils.

The oil composition, i.e., the properties and proportions of the fattyacid constituents of the glycerolipids, can also be manipulated bycombining biomass or oil from at least two distinct species ofmicroalgae. In some embodiments, at least two of the distinct species ofmicroalgae have different glycerolipid profiles. The distinct species ofmicroalgae can be cultured together or separately as described herein,preferably under heterotrophic conditions, to generate the respectiveoils. Different species of microalgae can contain different percentagesof distinct fatty acid constituents in the cell's glycerolipids.

Generally, Prototheca strains have very little or no fatty acids withthe chain length C8-C14. For example, Prototheca moriformis (UTEX 1435),Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442) andPrototheca zopfii (UTEX 1438) contains no (or undectable amounts) C8fatty acids, between 0-0.01% C10 fatty acids, between 0.03-2.1% C12fatty acids and between 1.0-1.7% C14 fatty acids.

In some cases, the Protheca strains containing a transgene encoding afatty acyl-ACP thioesterase that has activity towards fatty acyl-ACPsubstrate of chain lengths C8-10 has at least 0.3%, at least 0.8%, atleast 1.5% or more fatty acids of chain length C8 and at least 0.3%, atleast 1.0%, at least 3.0%, at least 5% or more fatty acids of chainlength C10. In other instances, the Prototheca strains containing atransgene encoding a fatty acyl-ACP thioesterase that has activitytowards fatty acyl-ACP substrate of chain length C12 has at least 3.0%,at least 5%, at least 7%, at least 10%, at least 13% or more fatty acidsof the chain length C12 and at least 1.5%, at least 2%, or at least 3%or more fatty acids of the chain length C14. In other cases, thePrototheca strains containing a transgene encoding a fatty acyl-ACPthioesterase that has activity towards fatty acyl-ACP substrate of chainlength C14 has at least 4.0%, at least 7%, at least 10%, at least 15%,at least 20%, at least 25% or more fatty acids of the chain length C14,and at least 0.4%, at least 1%, at least 1.5%, or more fatty acids ofthe chain length C12.

In non-limiting examples, the Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain length C8 and C10 has between 0.3-1.58%fatty acids of chain length C8 and between 0.35-6.76% fatty acids of thechain length C10. In other non-limiting examples, Prototheca strainscontaining a transgene encoding a fatty acyl-ACP thioesterase that hasactivity towards fatty acyl-ACP substrate of chain length C12 hasbetween 3.9-14.11% fatty acids of the chain length C12 and between1.95-3.05% fatty acids of the chain length C14. In other non-limitingexamples, Prototheca strains containing a transgene encoding a fattyacyl-ACP thioesterase that has activity towards fatty acyl-ACP substrateof chain length C14 has between 4.40-17.35% fatty acids of the chainlength C14 and between 0.4-1.83 Area % fatty acids of the chain lengthC12. In some cases, the Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrate of chain lengths between C8 and C14 have between3.5-20% medium chain (C8-C14) fatty acids. In some instances, keepingthe transgenic Prototheca strains under constant and high selectivepressure to retain exogenous genes is advantageous due to the increasein the desired fatty acid of a specific chain length. In a non-limitingexample, Example 5 demonstrates a two fold increase in C14 chain lengthfatty acids (more than 30% C8-C14 chain length fatty acids) when theculture of Prototheca moriformis containing a C14 preferringthioesterase exogenous gene is retained. High levels of exogenous generetention can also be achieved by inserting exogenous genes into thenuclear chromosomes of the cells using homologous recombination vectorsand methods disclosed herein. Recombinant cells containing exogenousgenes integrated into nuclear chromosomes are an object of theinvention.

Microalgal oil can also include other constituents produced by themicroalgae, or incorporated into the microalgal oil from the culturemedium. These other constituents can be present in varying amountdepending on the culture conditions used to culture the microalgae, thespecies of microalgae, the extraction method used to recover microalgaloil from the biomass and other factors that may affect microalgal oilcomposition. Non-limiting examples of such constituents includecarotenoids, present from 0.1-0.4 micrograms/ml, chlorophyll presentfrom 0-0.02 milligrams/kilogram of oil, gamma tocopherol present from0.4-0.6 milligrams/100 grams of oil, and total tocotrienols present from0.2-0.5 milligrams/gram of oil.

The other constituents can include, without limitation, phospholipids,tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene,beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin,alpha-cryptoxanthin and beta-crytoxanthin), and various organic orinorganic compounds.

In some cases, the oil extracted from Prototheca species comprises nomore than 0.02 mg/kg chlorophyll. In some cases, the oil extracted fromPrototheca species comprises no more than 0.4 mcg/ml total carotenoids.In some cases the Prototheca oil comprises between 0.40-0.60 milligramsof gamma tocopherol per 100 grams of oil. In other cases, the Protothecaoil comprises between 0.2-0.5 milligrams of total tocotrienols per gramof oil.

III. GENETIC ENGINEERING METHODS AND MATERIALS

The present invention provides methods and materials for genenticallymodifying Prototheca cells and recombinant host cells useful in themethods of the present invention, including but not limited torecombinant Prototheca moriformis, Prototheca zopfii, Protothecakrugani, and Prototheca stagnora host cells. The description of thesemethods and materials is divided into subsections for the convenience ofthe reader. In subsection 1, transformation methods are described. Insubsection 2, genetic engineering methods using homologous recombinationare described. In subsection 3, expression vectors and components aredescribed.

1. Engineering Methods—Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation (see Maruyama et al. (2004), BiotechnologyTechniques 8:821-826), glass bead transformation and silicon carbidewhisker transformation. Another method that can be used involves formingprotoplasts and using CaCl₂ and polyethylene glycol (PEG) to introducerecombinant DNA into microalgal cells (see Kim et al. (2002), Mar.Biotechnol. 4:63-73, which reports the use of this method for thetransformation of Chorella ellipsoidea). Co-transformation of microalgaecan be used to introduce two distinct vector molecules into a cellsimultaneously (see for example Protist 2004 December; 155(4):381-93).

Biolistic methods (see, for example, Sanford, Trends In Biotech. (1988)6:299 302, U.S. Pat. No. 4,945,050; electroporation (Fromm et al., Proc.Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); use of a laser beam,microinjection or any other method capable of introducing DNA into amicroalgae can also be used for transformation of a Prototheca cell.

2. Engineering Methods—Homologous Recombination

Homologous recombination is the ability of complementary DNA sequencesto align and exchange regions of homology. Transgenic DNA (“donor”)containing sequences homologous to the genomic sequences being targeted(“template”) is introduced into the organism and then undergoesrecombination into the genome at the site of the corresponding genomichomologous sequences. The mechanistic steps of this process, in mostcases, include: (1) pairing of homologous DNA segments; (2) introductionof double-stranded breaks into the donor DNA molecule; (3) invasion ofthe template DNA molecule by the free donor DNA ends followed by DNAsynthesis; and (4) resolution of double-strand break repair events thatresult in final recombination products.

The ability to carry out homologous recombination in a host organism hasmany practical implications for what can be carried out at the moleculargenetic level and is useful in the generation of an oleaginous microbethat can produced tailored oils. By its very nature homologousrecombination is a precise gene targeting event, hence, most transgeniclines generated with the same targeting sequence will be essentiallyidentical in terms of phenotype, necessitating the screening of farfewer transformation events. Homologous recombination also targets geneinsertion events into the host chromosome, resulting in excellentgenetic stability, even in the absence of genetic selection. Becausedifferent chromosomal loci will likely impact gene expression, even fromheterologous promoters/UTRs, homologous recombination can be a method ofquerying loci in an unfamiliar genome environment and to assess theimpact of these environments on gene expression.

Particularly useful genetic engineering applications using homologousrecombination is to co-opt specific host regulatory elements such aspromoters/UTRs to drive heterologous gene expression in a highlyspecific fashion. For example, precise ablation of the endogenousstearoyl ACP desaturase gene with a heterologous C12:0 specific FATB(thioesterase) gene cassette and suitable selective marker, might beexpected to dramatically decrease endogenous levels of C18:1 fatty acidsconcomitant with increased levels of the C12:0 fatty acids. Example 13describes the homologous recombination targeting construct that issuitable for the eblation of an endogenous Prototheca moriformisstearoyl ACP destaurase gene.

Because homologous recombination is a precise gene targeting event, itcan be used to precisely modify any nucleotide(s) within a gene orregion of interest, so long as sufficient flanking regions have beenidentified. Therefore, homologous recombination can be used as a meansto modify regulatory sequences impacting gene expression of RNA and/orproteins. It can also be used to modify protein coding regions in aneffort to modify enzyme activities such as substrate specificity,affinities and Km, and thus affecting the desired change in metabolismof the host cell. Homologous recombination provides a powerful means tomanipulate the gost genome resulting in gene targeting, gene conversion,gene deletion, gene duplication, gene inversion and exchanging geneexpression regulatory elements such as promoters, enhancers and 3′UTRs.

Homologous recombination can be achieve by using targeting constructscontaining pieces of endogenous sequences to “target” the gene or regionof interest within the endogenous host cell genome. Such targetingsequences can either be located 5′ of the gene or region of interest, 3′of the gene/region of interest or even flank the gene/region ofinterest. Such targeting constructs can be transformed into the hostcell either as a supercoiled plasmid DNA with additional vectorbackbone, a PCR product with no vector backbone, or as a linearizedmolecule. In some cases, it may be advantageous to first expose thehomologous sequences within the transgenic DNA (donor DNA) with arestriction enzyme. This step can increase the recombination efficiencyand decrease the occurrence of undesired events. Other methods ofincreasing recombination efficiency include using PCR to generatetransforming transgenic DNA containing linear ends homologous to thegenomic sequences being targeted.

3. Vectors and Vector Components

Vectors for transformation of microorganisms in accordance with thepresent invention can be prepared by known techniques familiar to thoseskilled in the art in view of the disclosure herein. A vector typicallycontains one or more genes, in which each gene codes for the expressionof a desired product (the gene product) and is operably linked to one ormore control sequences that regulate gene expression or target the geneproduct to a particular location in the recombinant cell. To aid thereader, this subsection is divided into subsections. Subsection Adescribes control sequences typically contained on vectors as well asnovel control sequences provided by the present invention. Subsection Bdescribes genes typically contained in vectors as well as novel codonoptimization methods and genes prepared using them provided by theinvention.

A. Control Sequences

Control sequences are nucleic acids that regulate the expression of acoding sequence or direct a gene product to a particular location in oroutside a cell. Control sequences that regulate expression include, forexample, promoters that regulate transcription of a coding sequence andterminators that terminate transcription of a coding sequence. Anothercontrol sequence is a 3′ untranslated sequence located at the end of acoding sequence that encodes a polyadenylation signal. Control sequencesthat direct gene products to particular locations include those thatencode signal peptides, which direct the protein to which they areattached to a particular location in or outside the cell.

Thus, an exemplary vector design for expression of an exogenous gene ina microalgae contains a coding sequence for a desired gene product (forexample, a selectable marker, a lipid pathway modification enzyme, or asucrose utilization enzyme) in operable linkage with a promoter activein microalgae. Alternatively, if the vector does not contain a promoterin operable linkage with the coding sequence of interest, the codingsequence can be transformed into the cells such that it becomes operablylinked to an endogenous promoter at the point of vector integration. Thepromoterless method of transformation has been proven to work inmicroalgae (see for example Plant Journal 14:4, (1998), pp. 441-447).

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Illustrative exogenous and/or endogenouspromoters that are active in microalgae (as well as antibioticresistance genes functional in microalgae) are described in PCT Pub. No.2008/151149 and references cited therein).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Illustrative promoters include promoterssuch as β-tubulin from Chlamydomonas reinhardtii, used in the Examplesbelow, and viral promoters, such as cauliflower mosaic virus (CMV) andchlorella virus, which have been shown to be active in multiple speciesof microalgae (see for example Plant Cell Rep. 2005 March;23(10-11):727-35; J Microbiol. 2005 August; 43(4):361-5; Mar Biotechnol(NY). 2002 January; 4(1):63-73). Another promoter that is suitable foruse for expression of exogenous genes in Prototheca is the Chlorellasorokiniana glutamate dehydrogenase promoter/5′UTR (SEQ ID NO: 69).Optionally, at least 10, 20, 30, 40, 50, or 60 nucleotides or more ofthese sequences containing a promoter are used. Illustrative promotersuseful for expression of exogenous genes in Prototheca are listed in thesequence listing of this application, such as the promoter of theChlorella HUP1 gene (SEQ ID NO:1) and the Chlorella ellipsoidea nitratereductase promoter (SEQ ID NO:2). Chlorella virus promoters can also beused to express genes in Prototheca, such as SEQ ID NOs: 1-7 of U.S.Pat. No. 6,395,965. Additional promoters active in Prototheca can befound, for example, in Biochem Biophys Res Commun. 1994 Oct. 14;204(1):187-94; Plant Mol Biol. 1994 October; 26(1):85-93; Virology. 2004Aug. 15; 326(1):150-9; and Virology. 2004 Jan. 5; 318(1):214-23.

A promoter can generally be characterized as either constitutive orinducible. Constitutive promoters are generally active or function todrive expression at all times (or at certain times in the cell lifecycle) at the same level. Inducible promoters, conversely, are active(or rendered inactive) or are significantly up- or down-regulated onlyin response to a stimulus. Both types of promoters find application inthe methods of the invention. Inducible promoters useful in theinvention include those that mediate transcription of an operably linkedgene in response to a stimulus, such as an exogenously provided smallmolecule (e.g, glucose, as in SEQ ID NO:1), temperature (heat or cold),lack of nitrogen in culture media, etc. Suitable promoters can activatetranscription of an essentially silent gene or upregulate, preferablysubstantially, transcription of an operably linked gene that istranscribed at a low level.

Inclusion of termination region control sequence is optional, and ifemployed, then the choice is be primarily one of convenience, as thetermination region is relatively interchangeable. The termination regionmay be native to the transcriptional initiation region (the promoter),may be native to the DNA sequence of interest, or may be obtainable fromanother source. See, for example, Chen and Orozco, Nucleic Acids Res.(1988) 16:8411.

The present invention also provides control sequences and recombinantgenes and vectors containing them that provide for the compartmentalizedexpression of a gene of interest. Organelles for targeting arechloroplasts, plastids, mitochondria, and endoplasmic reticulum. Inaddition, the present invention provides control sequences andrecombinant genes and vectors containing them that provide for thesecretion of a protein outside the cell.

Proteins expressed in the nuclear genome of Prototheca can be targetedto the plastid using plastid targeting signals. Plastid targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to theplastid; see for example GenBank Accession numbers AY646197 andAF499684, and in one embodiment, such control sequences are used in thevectors of the present invention to target expression of a protein to aPrototheca plastid.

The Examples below describe the use of algal plastid targeting sequencesto target heterologous proteins to the correct compartment in the hostcell. cDNA libraries were made using Prototheca moriformis and Chlorellaprotothecodies cells and are described in Examples 12 and Example 11below. Sequences were BLASTed and analyzed for homology to knownproteins that traffic to the plastid/chloroplast. The cDNAs encodingthese proteins were cloned and plastid targeting sequences were isolatedfrom these cDNAs. The amino acid sequences of the algal plastidtargeting sequences identified from the cDNA libraries and the aminoacid sequences of plant fatty acyl-ACP thioesterases that are used inthe heterologous expression Examples below are listed in SEQ ID NOs:127-133.

In another embodiment of the present invention, the expression of apolypeptide in Prototheca is targeted to the endoplasmic reticulum. Theinclusion of an appropriate retention or sorting signal in an expressionvector ensure that proteins are retained in the endoplasmic reticulum(ER) and do not go downstream into Golgi. For example, theIMPACTVECTOR1.3 vector, from Wageningen UR-Plant Research International,includes the well known KDEL retention or sorting signal. With thisvector, ER retention has a practical advantage in that it has beenreported to improve expression levels 5-fold or more. The main reasonfor this appears to be that the ER contains lower concentrations and/ordifferent proteases responsible for post-translational degradation ofexpressed proteins than are present in the cytoplasm. ER retentionsignals functional in green microalgae are known. For example, see ProcNatl Acad Sci USA. 2005 Apr. 26; 102(17):6225-30.

In another embodiment of the present invention, a polypeptide istargeted for secretion outside the cell into the culture media. SeeHawkins et al., Current Microbiology Vol. 38 (1999), pp. 335-341 forexamples of secretion signals active in Chlorella that can be used, inaccordance with the methods of the invention, in Prototheca.

B. Genes and Codon Optimization

Typically, a gene includes a promoter, coding sequence, and terminationcontrol sequences. When assembled by recombinant DNA technology, a genemay be termed an expression cassette and may be flanked by restrictionsites for convenient insertion into a vector that is used to introducethe recombinant gene into a host cell. The expression cassette can beflanked by DNA sequences from the genome or other nucleic acid target tofacilitate stable integration of the expression cassette into the genomeby homologous recombination. Alternatively, the vector and itsexpression cassette may remain unintegrated, in which case, the vectortypically includes an origin of replication, which is capable ofproviding for replication of the heterologous vector DNA.

A common gene present on a vector is a gene that codes for a protein,the expression of which allows the recombinant cell containing theprotein to be differentiated from cells that do not express the protein.Such a gene, and its corresponding gene product, is called a selectablemarker. Any of a wide variety of selectable markers can be employed in atransgene construct useful for transforming Prototheca. Examples ofsuitable selectable markers include the G418 resistance gene, thenitrate reductase gene (see Dawson et al. (1997), Current Microbiology35:356-362), the hygromycin phosphotransferase gene (HPT; see Kim et al.(2002), Mar. Biotechnol. 4:63-73), the neomycin phosphotransferase gene,and the ble gene, which confers resistance to phleomycin (Huang et al.(2007), Appl. Microbiol. Biotechnol. 72:197-205). Methods of determiningsensitivity of microalgae to antibiotics are well known. For example,Mol Gen Genet. 1996 Oct. 16; 252(5):572-9.

For purposes of the present invention, the expression vector used toprepare a recombinant host cell of the invention will include at leasttwo, and often three, genes, if one of the genes is a selectable marker.For example, a genetically engineered Prototheca of the invention can bemade by transformation with vectors of the invention that comprise, inaddition to a selectable marker, one or more exogenous genes, such as,for example, sucrose invertase gene or acyl ACP-thioesterase gene. Oneor both genes can be expressed using an inducible promoter, which allowsthe relative timing of expression of these genes to be controlled toenhance the lipid yield and conversion to fatty acid esters. Expressionof the two or more exogenous genes may be under control of the sameinducible promoter or under control of different inducible (orconstitutive) promoters. In the latter situation, expression of a firstexogenous gene can be induced for a first period of time (during whichexpression of a second exogenous gene may or may not be induced) andexpression of a second exogenous gene can be induced for a second periodof time (during which expression of a first exogenous gene may or maynot be induced).

In other embodiments, the two or more exogenous genes (in addition toany selectable marker) are: a fatty acyl-ACP thioesterase and a fattyacyl-CoA/aldehyde reductase, the combined action of which yields analcohol product. Further provided are other combinations of exogenousgenes, including without limitation, a fatty acyl-ACP thioesterase and afatty acyl-CoA reductase to generate aldehydes. In one embodiment, thevector provides for the combination of a fatty acyl-ACP thioesterase, afatty acyl-CoA reductase, and a fatty aldehyde decarbonylase to generatealkanes. In each of these embodiments, one or more of the exogenousgenes can be expressed using an inducible promoter.

Other illustrative vectors of the invention that express two or moreexogenous genes include those encoding both a sucrose transporter and asucrose invertase enzyme and those encoding both a selectable marker anda secreted sucrose invertase. The recombinant Prototheca transformedwith either type of vector produce lipids at lower manufacturing costdue to the engineered ability to use sugar cane (and sugar cane-derivedsugars) as a carbon source. Insertion of the two exogenous genesdescribed above can be combined with the disruption of polysaccharidebiosynthesis through directed and/or random mutagenesis, which steersever greater carbon flux into lipid production. Individually and incombination, trophic conversion, engineering to alter lipid productionand treatment with exogenous enzymes alter the lipid compositionproduced by a microorganism. The alteration can be a change in theamount of lipids produced, the amount of one or more hydrocarbon speciesproduced relative to other lipids, and/or the types of lipid speciesproduced in the microorganism. For example, microalgae can be engineeredto produce a higher amount and/or percentage of TAGs.

For optimal expression of a recombinant protein, it is beneficial toemploy coding sequences that produce mRNA with codons preferentiallyused by the host cell to be transformed. Thus, proper expression oftransgenes can require that the codon usage of the transgene matches thespecific codon bias of the organism in which the transgene is beingexpressed. The precise mechanisms underlying this effect are many, butinclude the proper balancing of available aminoacylated tRNA pools withproteins being synthesized in the cell, coupled with more efficienttranslation of the transgenic messenger RNA (mRNA) when this need ismet. When codon usage in the transgene is not optimized, available tRNApools are not sufficient to allow for efficient translation of theheterologous mRNA resulting in ribosomal stalling and termination andpossible instability of the transgenic mRNA.

The present invention provides codon-optimized nucleic acids useful forthe successful expression of recombinant proteins in Prototheca. Codonusage in Prototheca species was analyzed by studying cDNA sequencesisolated from Prototheca moriformis. This analysis represents theinterrogation over 24,000 codons and resulted in Table 1 below.

TABLE 1 Preferred codon usage in Prototheca strains. Ala GCG 345 (0.36)Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101 (0.11) Pro CCG 161(0.29) GCC 442 (0.46) CCA 49 (0.09) Cys TGT 12 (0.10) CCT 71 (0.13) TGC105 (0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316(0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33 (0.06) GAA 14 (0.04)AGA 14 (0.02) Phe TTT 89 (0.29) CGG 102 (0.18) TTC 216 (0.71) CGA 49(0.08) Gly GGG 92 (0.12) CGT 51 (0.09) GGA 56 (0.07) CGC 331 (0.57) GGT76 (0.10) Ser AGT 16 (0.03) GGC 559 (0.71) AGC 123 (0.22) His CAT 42(0.21) TCG 152 (0.28) CAC 154 (0.79) TCA 31 (0.06) Ile ATA 4 (0.01) TCT55 (0.10) ATT 30 (0.08) TCC 173 (0.31) ATC 338 (0.91) Thr ACG 184 (0.38)Lys AAG 284 (0.98) ACA 24 (0.05) AAA 7 (0.02) ACT 21 (0.05) Leu TTG 26(0.04) ACC 249 (0.52) TTA 3 (0.00) Val GTG 308 (0.50) CTG 447 (0.61) GTA9 (0.01) CTA 20 (0.03) GTT 35 (0.06) CTT 45 (0.06) GTC 262 (0.43) CTC190 (0.26) Trp TGG 107 (1.00) Met ATG 191 (1.00) Tyr TAT 10 (0.05) TAC180 (0.95) Stop TGA/TAG/TAA

In other embodiments, the gene in the recombinant vector has beencodon-optimized with reference to a microalgal strain other than aPrototheca strain. For example, methods of recoding genes for expressionin microalgae are described in U.S. Pat. No. 7,135,290. Additionalinformation for codon optimization is available, e.g., at the codonusage database of GenBank.

While the methods and materials of the invention allow for theintroduction of any exogenous gene into Prototheca, genes relating tosucrose utilization and lipid pathway modification are of particularinterest, as discussed in the following sections.

IV. SUCROSE UTILIZATION

In embodiment, the recombinant Prototheca cell of the invention furthercontains one or more exogenous sucrose utilization genes. In variousembodiments, the one or more genes encode one or more proteins selectedfrom the group consisting of a fructokinase, a glucokinase, ahexokinase, a sucrose invertase, a sucrose transporter. For example,expression of a sucrose transporter and a sucrose invertase allowsPrototheca to transport sucrose into the cell from the culture media andhydrolyze sucrose to yield glucose and fructose. Optionally, afructokinase can be expressed as well in instances where endogenoushexokinase activity is insufficient for maximum phosphorylation offructose. Examples of suitable sucrose transporters are Genbankaccession numbers CAD91334, CAB92307, and CAA53390. Examples of suitablefructokinases are Genbank accession numbers P26984, P26420 and CAA43322.

In one embodiment, the present invention provides a Prototheca host cellthat secretes a sucrose invertase. Secretion of a sucrose invertaseobviates the need for expression of a transporter that can transportsucrose into the cell. This is because a secreted invertase catalyzesthe conversion of a molecule of sucrose into a molecule of glucose and amolecule of fructose, both of which can be transported and utilized bymicrobes provided by the invention. For example, expression of a sucroseinvertase (such as SEQ ID NO:3) with a secretion signal (such as that ofSEQ ID NO: 4 (from yeast), SEQ ID NO: 5 (from higher plants), SEQ ID NO:6 (eukaryotic consensus secretion signal), and SEQ ID NO: 7 (combinationof signal sequence from higher plants and eukaryotic consensus)generates invertase activity outside the cell. Expression of such aprotein, as enabled by the genetic engineering methodology disclosedherein, allows cells already capable of utilizing extracellular glucoseas an energy source to utilize sucrose as an extracellular energysource.

Prototheca species expressing an invertase in media containing sucroseare a preferred microalgal species for the production of oil. Example 3illustrates how the methods and reagents of the invention can be used toexpress a recombinant yeast invertase and secrete it from a recombinantPrototheca cell. The expression and extracellular targeting of thisfully active protein allows the resulting host cells to grow on sucrose,whereas their non-transformed counterparts cannot. Thus, the presentinvention provides Prototheca recombinant cells with a codon-optimizedinvertase gene, including but not limited to the yeast invertase gene,integrated into their genome such that the invertase gene is expressedas assessed by invertase activity and sucrose hydrolysis. The presentinvention also provides invertase genes useful as selectable markers inPrototheca recombinant cells, as such cells are able to grow on sucrose,while their non-transformed counterparts cannot; and methods forselecting recombinant host cells using an invertase as a powerful,selectable marker for algal molecular genetics.

The successful expression of a sucrose invertase in Prototheca alsoillustrates another aspect of the present invention in that itdemonstrates that heterologous (recombinant) proteins can be expressedin the algal cell and successfully transit outside of the cell and intothe culture medium in a fully active and functional form. Thus, thepresent invention provides methods and reagents for expressing a wideand diverse array of heterologous proteins in microalgae and secretingthem outside of the host cell. Such proteins include, for example,industrial enzymes such as, for example, lipases, proteases, cellulases,pectinases, amylases, esterases, oxidoreductases, transferases,lactases, isomerases, and invertases, as well as therapeutic proteinssuch as, for example, growth factors, cytokines, full length antibodiescomprising two light and two heavy chains, Fabs, scFvs (single chainvariable fragment), camellid-type antibodies, antibody fragments,antibody fragment-fusions, antibody-receptor fusions, insulin,interferons, and insulin-like growth factors.

The successful expression of a sucrose invertase in Prototheca alsoillustrates another aspect of the present invention in that it providesmethods and reagents for the use of fungal transit peptides in algae todirect secretion of proteins in Prototheca; and methods and reagents fordetermining if a peptide can function, and the ability of it tofunction, as a transit peptide in Prototheca cells. The methods andreagents of the invention can be used as a tool and platform to identifyother transit peptides that can successfully traffic proteins outside ofa cell, and that the yeast invertase has great utility in these methods.As demonstrated in this example, removal of the endogenous yeastinvertase transit peptide and its replacement by other transit peptides,either endogenous to the host algae or from other sources (eukaryotic,prokaryotic and viral), can identify whether any peptide of interest canfunction as a transit peptide in guiding protein egress from the cell.

Examples of suitable sucrose invertases include those identified byGenbank accession numbers CAB95010, NP_012104 and CAA06839. Non-limitingexamples of suitable invertases are listed below in Table 2 Amino acidsequences for each listed invertase are included in the Sequence Listingbelow. In some cases, the exogenous sucrose utilization gene suitablefor use in the methods and vectors of the invention encodes a sucroseinvertase that has at least 40, 50, 60, 75, or 90% or higher amino acididentity with a sucrose invertase selected from Table 2.

TABLE 2 Sucrose invertases. Description Organism GenBank Accession No.SEQ ID NO: Invertase Chicorium intybus Y11124 SEQ ID NO: 20 InvertaseSchizosaccharomyces AB011433 SEQ ID NO: 21 pombe beta-fructofuranosidasePichia anomala X80640 SEQ ID NO: 22 (invertase) Invertase Debaryomycesoccidentalis X17604 SEQ ID NO: 23 Invertase Oryza sativa AF019113 SEQ IDNO: 24 Invertase Allium cepa AJ006067 SEQ ID NO: 25 Invertase Betavulgaris subsp. AJ278531 SEQ ID NO: 26 Vulgaris beta-fructofuranosidaseBifidobacterium breve AAT28190 SEQ ID NO: 27 (invertase) UCC2003Invertase Saccharomyces cerevisiae NP_012104 SEQ ID NO: 8 (nucleotide)SEQ ID NO: 28 (amino acid) Invertase A Zymomonas mobilis AAO38865 SEQ IDNO: 29

The secretion of an invertase to the culture medium by Prototheca enablethe cells to grow as well on waste molasses from sugar cane processingas they do on pure reagent-grade glucose; the use of this low-valuewaste product of sugar cane processing can provide significant costsavings in the production of lipids and other oils. Thus, the presentinvention provides a microbial culture containing a population ofPrototheca microorganisms, and a culture medium comprising (i) sucroseand (ii) a sucrose invertase enzyme. In various embodiments the sucrosein the culture comes from sorghum, sugar beet, sugar cane, molasses, ordepolymerized cellulosic material (which may optionally contain lignin).In another aspect, the methods and reagents of the inventionsignificantly increase the number and type of feedstocks that can beutilized by recombinant Prototheca. While the microbes exemplified hereare altered such that they can utilize sucrose, the methods and reagentsof the invention can be applied so that feedstocks such as cellulosicsare utilizable by an engineered host microbe of the invention with theability to secrete cellulases, pectinases, isomerases, or the like, suchthat the breakdown products of the enzymatic reactions are no longerjust simply tolerated but rather utilized as a carbon source by thehost.

V. LIPID PATHWAY ENGINEERING

In addition to altering the ability of Prototheca to utilize feedstockssuch as sucrose-containing feedstocks, the present invention alsoprovides recombinant Prototheca that have been modified to alter theproperties and/or proportions of lipids produced. The pathway canfurther, or alternatively, be modified to alter the properties and/orproportions of various lipid molecules produced through enzymaticprocessing of lipids and intermediates in the fatty acid pathway. Invarious embodiments, the recombinant Prototheca cells of the inventionhave, relative to their untransformed counterparts, optimized lipidyield per unit volume and/or per unit time, carbon chain length (e.g.,for renewable diesel production or for industrial chemicals applicationsrequiring lipid feedstock), reduced number of double or triple bonds,optionally to zero, and increasing the hydrogen:carbon ratio of aparticular species of lipid or of a population of distinct lipid.

In particular embodiments, one or more key enzymes that control branchpoints in metabolism to fatty acid synthesis have been up-regulated ordown-regulated to improve lipid production. Up-regulation can beachieved, for example, by transforming cells with expression constructsin which a gene encoding the enzyme of interest is expressed, e.g.,using a strong promoter and/or enhancer elements that increasetranscription. Such constructs can include a selectable marker such thatthe transformants can be subjected to selection, which can result inamplification of the construct and an increase in the expression levelof the encoded enzyme. Examples of enzymes suitable for up-regulationaccording to the methods of the invention include pyruvatedehydrogenase, which plays a role in converting pyruvate to acetyl-CoA(examples, some from microalgae, include Genbank accession numbersNP_415392; AAA53047; Q1XDM1; and CAF05587). Up-regulation of pyruvatedehydrogenase can increase production of acetyl-CoA, and therebyincrease fatty acid synthesis. Acetyl-CoA carboxylase catalyzes theinitial step in fatty acid synthesis. Accordingly, this enzyme can beup-regulated to increase production of fatty acids (examples, some frommicroalgae, include Genbank accession numbers BAA94752; AAA75528;AAA81471; YP_537052; YP_536879; NP_045833; and BAA57908). Fatty acidproduction can also be increased by up-regulation of acyl carrierprotein (ACP), which carries the growing acyl chains during fatty acidsynthesis (examples, some from microalgae, include Genbank accessionnumbers A0T0F8; P51280; NP_849041; YP_874433). Glycerol-3-phosphateacyltransferase catalyzes the rate-limiting step of fatty acidsynthesis. Up-regulation of this enzyme can increase fatty acidproduction (examples, some from microalgae, include Genbank accessionnumbers AAA74319; AAA33122; AAA37647; P44857; and ABO94442).

Up- and/or down-regulation of genes can be applied to global regulatorscontrolling the expression of the genes of the fatty acid biosyntheticpathways. Accordingly, one or more global regulators of fatty acidsynthesis can be up- or down-regulated, as appropriate, to inhibit orenhance, respectively, the expression of a plurality of fatty acidsynthetic genes and, ultimately, to increase lipid production. Examplesinclude sterol regulatory element binding proteins (SREBPs), such asSREBP-1a and SREBP-1c (for examples see Genbank accession numbersNP_035610 and Q9WTN3).

The present invention also provides recombinant Prototheca cells thathave been modified to contain one or more exogenous genes encoding lipidmodification enzymes such as, for example, fatty acyl-ACP thioesterases(see Table 3), fatty acyl-CoA/aldehyde reductases (see Table 4), fattyacyl-CoA reductases (see Table 5), fatty aldehyde decarbonylase (seeTable 6), fatty aldehyde reductases, and squalene synthases (see GenBankAccession number AF205791). In some embodiments, genes encoding a fattyacyl-ACP thioesterase and a naturally co-expressed acyl carrier proteinare transformed into a Prototheca cell, optionally with one or moregenes encoding other lipid modification enzymes. In other embodiments,the ACP and the fatty acyl-ACP thioesterase may have an affinity for oneanother that imparts an advantage when the two are used together in themicrobes and methods of the present invention, irrespective of whetherthey are or are not naturally co-expressed in a particular tissue ororganism. Thus, the present invention contemplates both naturallyco-expressed pairs of these enzymes as well as those that share anaffinity for interacting with one another to facilitate cleavage of alength-specific carbon chain from the ACP.

In still other embodiments, an exogenous gene encoding a desaturase istransformed into the Prototheca cell in conjunction with one or moregenes encoding other lipid modification enzymes to provide modificationswith respect to lipid saturation. Stearoyl-ACP desaturase (see, e.g.,GenBank Accession numbers AAF15308; ABM45911; and AAY86086), forexample, catalyzes the conversion of stearoyl-ACP to oleoyl-ACP.Up-regulation of this gene can increase the proportion ofmonounsaturated fatty acids produced by a cell; whereas down-regulationcan reduce the proportion of monounsaturates. Similarly, the expressionof one or more glycerolipid desaturases can be controlled to alter theratio of unsaturated to saturated fatty acids such as ω-6 fatty aciddesaturase, ω-3 fatty acid desaturase, or ω-6-oleate desaturase. In someembodiments, the desaturase can be selected with reference to a desiredcarbon chain length, such that the desaturase is capable of makinglocation specific modifications within a specified carbon-lengthsubstrate, or substrates having a carbon-length within a specifiedrange.

Thus, in particular embodiments, microbes of the present invention aregenetically engineered to express one or more exogenous genes selectedfrom an acyl-ACP thioesterase, an acyl-CoA/aldehyde reductase, a fattyacyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehydedecarbonylase, or a naturally co-expressed acyl carrier protein.Suitable expression methods are described above with respect to theexpression of a lipase gene, including, among other methods, inducibleexpression and compartmentalized expression. A fatty acyl-ACPthioesterase cleaves a fatty acid from an acyl carrier protein (ACP)during lipid synthesis. Through further enzymatic processing, thecleaved fatty acid is then combined with a coenzyme to yield an acyl-CoAmolecule. This acyl-CoA is the substrate for the enzymatic activity of afatty acyl-CoA reductase to yield an aldehyde, as well as for a fattyacyl-CoA/aldehyde reductase to yield an alcohol. The aldehyde producedby the action of the fatty acyl-CoA reductase identified above is thesubstrate for further enzymatic activity by either a fatty aldehydereductase to yield an alcohol, or a fatty aldehyde decarbonylase toyield an alkane or alkene.

In some embodiments, fatty acids, glycerolipids, or the correspondingprimary alcohols, aldehydes, alkanes or alkenes, generated by themethods described herein, contain 8, 10, 12, or 14 carbon atoms.Preferred fatty acids for the production of diesel, biodiesel, renewablediesel, or jet fuel, or the corresponding primary alcohols, aldehydes,alkanes and alkenes, for industrial applications contain 8 to 14 carbonatoms. In certain embodiments, the above fatty acids, as well as theother corresponding hydrocarbon molecules, are saturated (with nocarbon-carbon double or triple bonds); mono unsaturated (single doublebond); poly unsturated (two or more double bonds); are linear (notcyclic) or branched. For fuel production, greater saturation ispreferred.

The enzymes described directly above have a preferential specificity forhydrolysis of a substrate containing a specific number of carbon atoms.For example, a fatty acyl-ACP thioesterase may have a preference forcleaving a fatty acid having 12 carbon atoms from the ACP. In someembodiments, the ACP and the length-specific thioesterase may have anaffinity for one another that makes them particularly useful as acombination (e.g., the exogenous ACP and thioesterase genes may benaturally co-expressed in a particular tissue or organism from whichthey are derived). Therefore, in various embodiments, the recombinantPrototheca cell of the invention can contain an exogenous gene thatencodes a protein with specificity for catalyzing an enzymatic activity(e.g., cleavage of a fatty acid from an ACP, reduction of an acyl-CoA toan aldehyde or an alcohol, or conversion of an aldehyde to an alkane)with regard to the number of carbon atoms contained in the substrate.The enzymatic specificity can, in various embodiments, be for asubstrate having from 8 to 34 carbon atoms, preferably from 8 to 18carbon atoms, and more preferably from 8 to 14 carbon atoms. A preferredspecificity is for a substrate having fewer, i.e., 12, rather than more,i.e., 18, carbon atoms.

In non-limiting but illustrative examples, the present inventionprovides vectors and Prototheca host cells that express an exogenousthioesterase and accordingly produce lipid enriched, relative to thelipid profile of untransformed Prototheca cells, in the chain length forwhich the thioesterase is specific. The thioesterases illustrated are(i) Cinnamomum camphorum FatB1 (GenBank Accession No. Q39473, amino acidsequence is in SEQ ID NO: 59, amino acid sequence without plastidtargeting sequence (PTS) is in SEQ ID NO: 139, and codon optimized cDNAsequence based on Table 1 is in SEQ ID NO: 60), which has a preferencefor fatty acyl-ACP substrate with a carbon chain length of 14; (ii)Cuphea hookeriana FatB2 (GenBank Accession No. AAC49269, amino acidsequence is in SEQ ID NO: 61, amino acid sequence without PTS is in SEQID NO: 138, and codon optimized cDNA sequence based on Table 1 is in SEQID NO: 62), which has a preference for a fatty acyl-ACP substrate with acarbon chain length of 8-10; and (iii) Umbellularia Fat B1 (GenBankAccession No. Q41635, amino acid sequence is included in SEQ ID NO: 63,amino acid sequence without PTS is in SEQ ID NO: 139, and codonoptimized cDNA sequence based on Table 1 is included in SEQ ID NO: 64),which has a preference for a fatty acyl-ACP substrate with a carbonchain length of 12.

Other fatty acyl-ACP thioesterases suitable for use with the microbesand methods of the invention include, without limitation, those listedin Table 3.

TABLE 3 Fatty acyl-ACP thioesterases and GenBank accession numbers.Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001)Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #Q39473)Umbellularia californica fatty acyl-ACP thioesterase (GenBank #Q41635)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71729)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71730)Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #ABD83939) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAD42220) Populustomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #NP_172327) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85387) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85388) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3) Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank #CAA54060) Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank #AAC72882) Cuphea calophylla subsp.mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581) Cuphealanceolata fatty acyl-ACP thioesterase (GenBank #CAC19933) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAL15645) Cupheahookeriana fatty acyl-ACP thioesterase (GenBank #Q39513) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #AAD01982) Vitis viniferafatty acyl-ACP thioesterase (GenBank #CAN81819) Garcinia mangostanafatty acyl-ACP thioesterase (GenBank #AAB51525) Brassica juncea fattyacyl-ACP thioesterase (GenBank #ABI18986) Madhuca longifolia fattyacyl-ACP thioesterase (GenBank #AAX51637) Brassica napus fatty acyl-ACPthioesterase (GenBank #ABH11710) Oryza sativa (indica cultivar-group)fatty acyl-ACP thioesterase (GenBank #EAY86877) Oryza sativa (japonicacultivar-group) fatty acyl-ACP thioesterase (GenBank #NP_001068400)Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase(GenBank #EAY99617) Cuphea hookeriana fatty acyl-ACP thioesterase(GenBank #AAC49269) Ulmus Americana fatty acyl-ACP thioesterase (GenBank#AAB71731) Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank#CAB60830) Cuphea palustris fatty acyl-ACP thioesterase (GenBank#AAC49180) Iris germanica fatty acyl-ACP thioesterase (GenBank#AAG43858) Cuphea palustris fatty acyl-ACP thioesterase (GenBank#AAC49179) Myristica fragrans fatty acyl-ACP thioesterase(GenBank#AAB71729) Cuphea hookeriana fatty acyl-ACP thioesterase(GenBank #U39834) Umbelluaria californica fatty acyl-ACP thioesterase(GenBank #M94159) Cinnamomum camphora fatty acyl-ACP thioesterase(GenBank #U31813)

The Examples below describe the successful targeting and expression ofheterologous fatty acyl-ACP thioesterases from Cuphea hookeriana,Umbellularia californica, Cinnamomun camphora in Prototheca species.Additionally, alterations in fatty acid profiles were confirmed in thehost cells expression these heterologous fatty acyl-ACP thioesterases.These results were quite unexpected given the lack of sequence identitybetween algal and higher plant thioesterases in general, and betweenPrototheca moriformis fatty acyl-ACP thioesterase and the above listedheterologous fatty acyl-ACP thioesterases. Two Prototheca moriformisacyl-ACP thioesterases were isolated and sequenced. The sequences of thetwo cDNAs showed a high degree of identity between each other, differingin only 12 positions at the nucleotide level and five positions at theamino acid level, four of these in the plastid transit peptide. Furtheranalysis of genomic sequence from Prototheca moriformis confirmed thatthese two cDNAs were indeed encoded on separate contigs, and althoughhighly homolous, are encoded by two distinct genes. The cDNA and aminoacid sequence of the two Prototheca moriformis fatty acyl-ACPthioesterase, P. moriformis fatty acyl-ACP thioesterase-1 and P.moriformis fatty acyl-ACP thioesterase-2, are listed as SEQ ID NOs:134-137.

When the amino acid sequences of these two cDNAs were BLASTed againstthe NCBI database, the two most homologous sequences were fatty acyl-ACPthioesterases from Chlamydomonas reinhardtii and Arabidopsis thaliana.Surprisingly, the level of amino acid identity between the Protothecamoriformis fatty acyl-ACP thioesterases and higher plant thioesteraseswas fairly low, at only 49 and 37% identity. In addition, there also isa subtle difference in the sequences surrounding the amino terminalportion of the catalytic triad (NXHX₃₆C) among these fatty acyl-ACPthioesterases. Thirty nine of forty higher plant fatty acyl-ACPthioesterases surveyed showed the sequence LDMNQH surrounding the N andH residues at the amino terminus of the triad, while all of the algalsequences identified had the sequence MDMNGH. Given the low amino acidsequence identity and the differences surrounding the catalytic triad ofthe thioesterases, the successful results of expression of exogenousfatty acyl-ACP thioesterases obtained and described in the Examples wereunexpected, particularly given the fact that activity of the exogenousfatty acyl-ACP thioesterases was dependent on a functionalprotein-protein interaction with the endogenous Prototheca acyl carrierprotein.

Fatty acyl-CoA/aldehyde reductases suitable for use with the microbesand methods of the invention include, without limitation, those listedin Table 4.

TABLE 4 Fatty acyl-CoA/aldehyde reductases listed by GenBank accessionnumbers. AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344,YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335,ZP_01892096, ZP_01103974, ZP_01915077, YP_924106, YP_130411,ZP_01222731, YP_550815, YP_983712, YP_001019688, YP_524762, YP_856798,ZP_01115500, YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156,YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936, AAR88762,ABK28586, NP_197634, CAD30694, NP_001063962, BAD46254, NP_001030809,EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541,CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190,CAA52019, and BAC84377

Fatty acyl-CoA reductases suitable for use with the microbes and methodsof the invention include, without limitation, those listed in Table 5.

TABLE 5 Fatty acyl-CoA reductases listed by GenBank accession numbers.NP_187805, AB014927, NP_001049083, CAN83375, NP_191229, EAZ42242,EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280,NP_197642, NP_190041, AAL15288, and NP_190042

Fatty aldehyde decarbonylases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 6.

TABLE 6 Fatty aldehyde decarbonylases listed by GenBank accessionnumbers. NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825, EAY86731,CAL55686, XP_001420263, EAZ23849, NP_200588, NP_001063227, CAN83072,AAR90847, and AAR97643

Combinations of naturally co-expressed fatty acyl-ACP thioesterases andacyl carrier proteins are suitable for use with the microbes and methodsof the invention.

Additional examples of hydrocarbon or lipid modification enzymes includeamino acid sequences contained in, referenced in, or encoded by nucleicacid sequences contained or referenced in, any of the following U.S.Pat. Nos. 6,610,527; 6,451,576; 6,429,014; 6,342,380; 6,265,639;6,194,185; 6,114,160; 6,083,731; 6,043,072; 5,994,114; 5,891,697;5,871,988; 6,265,639, and further described in GenBank Accessionnumbers: AA018435; ZP_00513891; Q38710; AAK60613; AAK60610; AAK60611;NP_113747; CAB75874; AAK60612; AAF20201; BAA11024; AF205791; andCAA03710.

Other suitable enzymes for use with the microbes and the methods of theinvention include those that have at least 70% amino acid identity withone of the proteins listed in Tables 3-6, and that exhibit thecorresponding desired enzymatic activity (e.g., cleavage of a fatty acidfrom an acyl carrier protein, reduction of an acyl-CoA to an aldehyde oran alcohol, or conversion of an aldehyde to an alkane). In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth.

By selecting the desired combination of exogenous genes to be expressed,one can tailor the product generated by the microbe, which may then beextracted from the aqueous biomass. For example, the microbe cancontain: (i) an exogenous gene encoding a fatty acyl-ACP thioesterase;and, optionally, (ii) a naturally co-expressed acyl carrier protein oran acyl carrier protein otherwise having affinity for the fatty acyl-ACPthioesterase (or conversely); and, optionally, (iii) an exogenous geneencoding a fatty acyl-CoA/aldehyde reductase or a fatty acyl-CoAreductase; and, optionally, (iv) an exogenous gene encoding a fattyaldehyde reductase or a fatty aldehyde decarbonylase. The microbe, underculture conditions described herein, synthesizes a fatty acid linked toan ACP and the fatty acyl-ACP thioesterase catalyzes the cleavage of thefatty acid from the ACP to yield, through further enzymatic processing,a fatty acyl-CoA molecule. When present, the fatty acyl-CoA/aldehydereducatase catalyzes the reduction of the acyl-CoA to an alcohol.Similarly, the fatty acyl-CoA reductase, when present, catalyzes thereduction of the acyl-CoA to an aldehyde. In those embodiments in whichan exogenous gene encoding a fatty acyl-CoA reductase is present andexpressed to yield an aldehyde product, a fatty aldehyde reductase,encoded by the third exogenous gene, catalyzes the reduction of thealdehyde to an alcohol. Similarly, a fatty aldehyde decarbonylasecatalyzes the conversion of the aldehyde to an alkane or an alkene, whenpresent.

Genes encoding such enzymes can be obtained from cells already known toexhibit significant lipid production such as Chlorella protothecoides.Genes already known to have a role in lipid production, e.g., a geneencoding an enzyme that saturates double bonds, can be transformedindividually into recipient cells. However, to practice the invention itis not necessary to make a priori assumptions as to which genes arerequired. Methods for identifying genes that can alter (improve) lipidproduction in microalgae are described in PCT Pub. No. 2008/151149.

Thus, the present invention provides a Prototheca cell that has beengenetically engineered to express a lipid pathway enzyme at an alteredlevel compared to a wild-type cell of the same species. In some cases,the cell produces more lipid compared to the wild-type cell when bothcells are grown under the same conditions. In some cases, the cell hasbeen genetically engineered and/or selected to express a lipid pathwayenzyme at a higher level than the wild-type cell. In some cases, thelipid pathway enzyme is selected from the group consisting of pyruvatedehydrogenase, acetyl-CoA carboxylase, acyl carrier protein, andglycerol-3 phosphate acyltransferase. In some cases, the cell has beengenetically engineered and/or selected to express a lipid pathway enzymeat a lower level than the wild-type cell. In at least one embodiment inwhich the cell expresses the lipid pathway enzyme at a lower level, thelipid pathway enzyme comprises citrate synthase.

In some embodiments, the cell has been genetically engineered and/orselected to express a global regulator of fatty acid synthesis at analtered level compared to the wild-type cell, whereby the expressionlevels of a plurality of fatty acid synthetic genes are altered comparedto the wild-type cell. In some cases, the lipid pathway enzyme comprisesan enzyme that modifies a fatty acid. In some cases, the lipid pathwayenzyme is selected from a stearoyl-ACP desaturase and a glycerolipiddesaturase.

In other embodiments, the present invention is directed to anoil-producing microbe containing one or more exogenous genes, whereinthe exogenous genes encode protein(s) selected from the group consistingof a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, a fattyaldehyde reductase, a fatty acyl-CoA/aldehyde reductase, a fattyaldehyde decarbonylase, and an acyl carrier protein. In one embodiment,the exogenous gene is in operable linkage with a promoter, which isinducible or repressible in response to a stimulus. In some cases, thestimulus is selected from the group consisting of an exogenouslyprovided small molecule, heat, cold, and limited or no nitrogen in theculture media. In some cases, the exogenous gene is expressed in acellular compartment. In some embodiments, the cellular compartment isselected from the group consisting of a chloroplast, a plastid and amitochondrion. In some embodiments the microbe is Prototheca moriformis,Prototheca krugani, Prototheca stagnora or Prototheca zopfii.

In one embodiment, the exogenous gene encodes a fatty acid acyl-ACPthioesterase. In some cases, the thioesterase encoded by the exogenousgene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an acylcarrier protein (ACP). In some cases, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acidfrom an ACP. In one embodiment, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 12-carbon fatty acid from anACP.

In one embodiment, the exogenous gene encodes a fatty acyl-CoA/aldehydereductase. In some cases, the reductase encoded by the exogenous genecatalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to acorresponding primary alcohol. In some cases, the reductase encoded bythe exogenous gene catalyzes the reduction of a 10 to 14-carbon fattyacyl-CoA to a corresponding primary alcohol. In one embodiment, thereductase encoded by the exogenous gene catalyzes the reduction of a12-carbon fatty acyl-CoA to dodecanol.

The present invention also provides a recombinant Prototheca cellcontaining two exogenous genes, wherein a first exogenous gene encodes afatty acyl-ACP thioesterase and a second exogenous gene encodes aprotein selected from the group consisting of a fatty acyl-CoAreductase, a fatty acyl-CoA/aldehyde reductase, and an acyl carrierprotein. In some cases, the two exogenous genes are each in operablelinkage with a promoter, which is inducible in response to a stimulus.In some cases, each promoter is inducible in response to an identicalstimulus, such as limited or no nitrogen in the culture media.Limitation or complete lack of nitrogen in the culture media stimulatesoil production in some microorganisms such as Prototheca species, andcan be used as a trigger to induce oil production to high levels. Whenused in combination with the genetic engineering methods disclosedherein, the lipid as a percentage of dry cell weight can be pushed tohigh levels such as at least 30%, at least 40%, at least 50%, at least60%, at least 70% and at least 75%; methods disclosed herein provide forcells with these levels of lipid, wherein the lipid is at least 4%C8-C14, at least 0.3% C8, at least 2% C10, at least 2% C12, and at least2% C14. In some embodiments the cells are over 25% lipid by dry cellweight and contain lipid that is at least 10% C8-C14, at least 20%C8-C14, at least 30% C8-C14, 10-30% C8-C14 and 20-30% C8-C14.

The novel oils disclosed herein are distinct from other naturallyoccurring oils that are high in mic-chain fatty acids, such as palm oil,palm kernel oil, and coconut oil. For example, levels of contaminantssuch as carotenoids are far higher in palm oil and palm kernel oil thanin the oils of the invention. Palm and palm kernel oils in particularcontain alpha and beta carotenes and lycopene in much higher amountsthan is in the oils of the invention. In addition, over 20 differentcarotenoids are found in palm and palm kernel oil, whereas the Examplesdemonstrate that the oils of the invention contain very few carotenoidsspecies and very low levels. In addition, the levels of vitamin Ecompounds such as tocotrienols are far higher in palm, palm kernel, andcoconut oil than in the oils of the invention.

In one embodiment, the thioesterase encoded by the first exogenous genecatalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP. Insome embodiments, the second exogenous gene encodes a fattyacyl-CoA/aldehyde reductase which catalyzes the reduction of an 8 to18-carbon fatty acyl-CoA to a corresponding primary alcohol. In somecases, the thioesterase encoded by the first exogenous gene catalyzesthe cleavage of a 10 to 14-carbon fatty acid from an ACP, and thereductase encoded by the second exogenous gene catalyzes the reductionof a 10 to 14-carbon fatty acyl-CoA to the corresponding primaryalcohol, wherein the thioesterase and the reductase act on the samecarbon chain length. In one embodiment, the thioesterase encoded by thefirst exogenous gene catalyzes the cleavage of a 12-carbon fatty acidfrom an ACP, and the reductase encoded by the second exogenous genecatalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. Insome embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase which catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding aldehyde. In some embodiments, the secondexogenous gene encodes an acyl carrier protein that is naturallyco-expressed with the fatty acyl-ACP thioesterase.

In some embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase, and the microbe further contains a third exogenous geneencoding a fatty aldehyde decarbonylase. In some cases, the thioesteraseencoded by the first exogenous gene catalyzes the cleavage of an 8 to18-carbon fatty acid from an ACP, the reductase encoded by the secondexogenous gene catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding fatty aldehyde, and the decarbonylaseencoded by the third exogenous gene catalyzes the conversion of an 8 to18-carbon fatty aldehyde to a corresponding alkane, wherein thethioesterase, the reductase, and the decarbonylase act on the samecarbon chain length.

In some embodiments, the second exogenous gene encodes an acyl carrierprotein, and the microbe further contains a third exogenous geneencoding a protein selected from the group consisting of a fattyacyl-CoA reductase and a fatty acyl-CoA/aldehyde reductase. In somecases, the third exogenous gene encodes a fatty acyl-CoA reductase, andthe microbe further contains a fourth exogenous gene encoding a fattyaldehyde decarbonylase.

The present invention also provides methods for producing an alcoholcomprising culturing a population of recombinant Prototheca cells in aculture medium, wherein the cells contain (i) a first exogenous geneencoding a fatty acyl-ACP thioesterase, and (ii) a second exogenous geneencoding a fatty acyl-CoA/aldehyde reductase, and the cells synthesize afatty acid linked to an acyl carrier protein (ACP), the fatty acyl-ACPthioesterase catalyzes the cleavage of the fatty acid from the ACP toyield, through further processing, a fatty acyl-CoA, and the fattyacyl-CoA/aldehyde reductase catalyzes the reduction of the acyl-CoA toan alcohol.

The present invention also provides methods of producing a lipidmolecule in a Prototheca cell. In one embodiment, the method comprisesculturing a population of Prototheca cells in a culture medium, whereinthe cells contain (i) a first exogenous gene encoding a fatty acyl-ACPthioesterase, and (ii) a second exogenous gene encoding a fatty acyl-CoAreductase, and wherein the microbes synthesize a fatty acid linked to anacyl carrier protein (ACP), the fatty acyl-ACP thioesterase catalyzesthe cleavage of the fatty acid from the ACP to yield, through furtherprocessing, a fatty acyl-CoA, and the fatty acyl-CoA reductase catalyzesthe reduction of the acyl-CoA to an aldehyde.

The present invention also provides methods of producing a fatty acidmolecule having a specified carbon chain length in a Prototheca cell. Inone embodiment, the method comprises culturing a population oflipid-producing Prototheca cells in a culture medium, wherein themicrobes contain an exogenous gene encoding a fatty acyl-ACPthioesterase having an activity specific or preferential to a certaincarbon chain length, such as 8, 10, 12 or 14 carbon atoms, and whereinthe microbes synthesize a fatty acid linked to an acyl carrier protein(ACP) and the thioesterase catalyzes the cleavage of the fatty acid fromthe ACP when the fatty acid has been synthesized to the specific carbonchain length.

In the various embodiments described above, the Prototheca cell cancontain at least one exogenous gene encoding a lipid pathway enzyme. Insome cases, the lipid pathway enzyme is selected from the groupconsisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, apyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrierprotein, and a glycerol-3 phosphate acyltransferase. In other cases, thePrototheca cell contains a lipid modification enzyme selected from thegroup consisting of a fatty acyl-ACP thioesterase, a fattyacyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fattyaldehyde reductase, a fatty aldehyde decarbonylase, and/or an acylcarrier protein.

VI. FUELS AND CHEMICALS PRODUCTION

For the production of fuel in accordance with the methods of theinvention lipids produced by cells of the invention are harvested, orotherwise collected, by any convenient means. Lipids can be isolated bywhole cell extraction. The cells are first disrupted, and thenintracellular and cell membrane/cell wall-associated lipids as well asextracellular hydrocarbons can be separated from the cell mass, such asby use of centrifugation as described above. Intracellular lipidsproduced in microorganisms are, in some embodiments, extracted afterlysing the cells of the microorganism. Once extracted, the lipids arefurther refined to produce oils, fuels, or oleochemicals.

After completion of culturing, the microorganisms can be separated fromthe fermentation broth. Optionally, the separation is effected bycentrifugation to generate a concentrated paste. Centrifugation does notremove significant amounts of intracellular water from themicroorganisms and is not a drying step. The biomass can then optionallybe washed with a washing solution (e.g., DI water) to get rid of thefermentation broth and debris. Optionally, the washed microbial biomassmay also be dried (oven dried, lyophilized, etc.) prior to celldisruption. Alternatively, cells can be lysed without separation fromsome or all of the fermentation broth when the fermentation is complete.For example, the cells can be at a ratio of less than 1:1 v:v cells toextracellular liquid when the cells are lysed.

Microorganisms containing a lipid can be lysed to produce a lysate. Asdetailed herein, the step of lysing a microorganism (also referred to ascell lysis) can be achieved by any convenient means, includingheat-induced lysis, adding a base, adding an acid, using enzymes such asproteases and polysaccharide degradation enzymes such as amylases, usingultrasound, mechanical lysis, using osmotic shock, infection with alytic virus, and/or expression of one or more lytic genes. Lysis isperformed to release intracellular molecules which have been produced bythe microorganism. Each of these methods for lysing a microorganism canbe used as a single method or in combination simultaneously orsequentially. The extent of cell disruption can be observed bymicroscopic analysis. Using one or more of the methods described herein,typically more than 70% cell breakage is observed. Preferably, cellbreakage is more than 80%, more preferably more than 90% and mostpreferred about 100%.

In particular embodiments, the microorganism is lysed after growth, forexample to increase the exposure of cellular lipid and/or hydrocarbonfor extraction or further processing. The timing of lipase expression(e.g., via an inducible promoter) or cell lysis can be adjusted tooptimize the yield of lipids and/or hydrocarbons. Below are described anumber of lysis techniques. These techniques can be used individually orin combination.

In one embodiment of the present invention, the step of lysing amicroorganism comprises heating of a cellular suspension containing themicroorganism. In this embodiment, the fermentation broth containing themicroorganisms (or a suspension of microorganisms isolated from thefermentation broth) is heated until the microorganisms, i.e., the cellwalls and membranes of microorganisms degrade or breakdown. Typically,temperatures applied are at least 50° C. Higher temperatures, such as,at least 30° C. at least 60° C., at least 70° C., at least 80° C., atleast 90° C., at least 100° C., at least 110° C., at least 120° C., atleast 130° C. or higher are used for more efficient cell lysis. Lysingcells by heat treatment can be performed by boiling the microorganism.Alternatively, heat treatment (without boiling) can be performed in anautoclave. The heat treated lysate may be cooled for further treatment.Cell disruption can also be performed by steam treatment, i.e., throughaddition of pressurized steam. Steam treatment of microalgae for celldisruption is described, for example, in U.S. Pat. No. 6,750,048. Insome embodiments, steam treatment may be achieved by sparging steam intothe fermentor and maintaining the broth at a desired temperature forless than about 90 minutes, preferably less than about 60 minutes, andmore preferably less than about 30 minutes.

In another embodiment of the present invention, the step of lysing amicroorganism comprises adding a base to a cellular suspensioncontaining the microorganism. The base should be strong enough tohydrolyze at least a portion of the proteinaceous compounds of themicroorganisms used. Bases which are useful for solubilizing proteinsare known in the art of chemistry. Exemplary bases which are useful inthe methods of the present invention include, but are not limited to,hydroxides, carbonates and bicarbonates of lithium, sodium, potassium,calcium, and mixtures thereof. A preferred base is KOH. Base treatmentof microalgae for cell disruption is described, for example, in U.S.Pat. No. 6,750,048.

In another embodiment of the present invention, the step of lysing amicroorganism comprises adding an acid to a cellular suspensioncontaining the microorganism. Acid lysis can be effected using an acidat a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis ispreferably performed at above room temperature (e.g., at 40-160°, andpreferably a temperature of 50-130°. For moderate temperatures (e.g.,room temperature to 100° C. and particularly room temperature to 65°,acid treatment can usefully be combined with sonication or other celldisruption methods.

In another embodiment of the present invention, the step of lysing amicroorganism comprises lysing the microorganism by using an enzyme.Preferred enzymes for lysing a microorganism are proteases andpolysaccharide-degrading enzymes such as hemicellulase (e.g.,hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, Mo.;#H2125), pectinase (e.g., pectinase from Rhizopus sp.; Sigma Aldrich,St. Louis, Mo.; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g.,cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, Mo.;#C9422), and driselase (e.g., driselase from Basidiomycetes sp.; SigmaAldrich, St. Louis, Mo.; #D9515.

In other embodiments of the present invention, lysis is accomplishedusing an enzyme such as, for example, a cellulase such as apolysaccharide-degrading enzyme, optionally from Chlorella or aChlorella virus, or a proteases, such as Streptomyces griseus protease,chymotrypsin, proteinase K, proteases listed in Degradation ofPolylactide by Commercial Proteases, Oda Y et al., Journal of Polymersand the Environment, Volume 8, Number 1, January 2000, pp. 29-32(4),Alcalase 2.4 FG (Novozymes), and Flavourzyme 100 L (Novozymes). Anycombination of a protease and a polysaccharide-degrading enzyme can alsobe used, including any combination of the preceding proteases andpolysaccharide-degrading enzymes.

In another embodiment, lysis can be performed using an expeller press.In this process, biomass is forced through a screw-type device at highpressure, lysing the cells and causing the intracellular lipid to bereleased and separated from the protein and fiber (and other components)in the cell.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by using ultrasound, i.e., sonication. Thus,cells can also by lysed with high frequency sound. The sound can beproduced electronically and transported through a metallic tip to anappropriately concentrated cellular suspension. This sonication (orultrasonication) disrupts cellular integrity based on the creation ofcavities in cell suspension.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by mechanical lysis. Cells can be lysedmechanically and optionally homogenized to facilitate hydrocarbon (e.g.,lipid) collection. For example, a pressure disrupter can be used to pumpa cell containing slurry through a restricted orifice valve. Highpressure (up to 1500 bar) is applied, followed by an instant expansionthrough an exiting nozzle. Cell disruption is accomplished by threedifferent mechanisms: impingement on the valve, high liquid shear in theorifice, and sudden pressure drop upon discharge, causing an explosionof the cell. The method releases intracellular molecules. Alternatively,a ball mill can be used. In a ball mill, cells are agitated insuspension with small abrasive particles, such as beads. Cells breakbecause of shear forces, grinding between beads, and collisions withbeads. The beads disrupt the cells to release cellular contents. Cellscan also be disrupted by shear forces, such as with the use of blending(such as with a high speed or Waring blender as examples), the frenchpress, or even centrifugation in case of weak cell walls, to disruptcells.

In another embodiment of the present invention, the step of lysing amicroorganism is performed by applying an osmotic shock.

In another embodiment of the present invention, the step of lysing amicroorganism comprises infection of the microorganism with a lyticvirus. A wide variety of viruses are known to lyse microorganismssuitable for use in the present invention, and the selection and use ofa particular lytic virus for a particular microorganism is within thelevel of skill in the art. For example, paramecium bursaria chlorellavirus (PBCV-1) is the prototype of a group (family Phycodnaviridae,genus Chlorovirus) of large, icosahedral, plaque-forming,double-stranded DNA viruses that replicate in, and lyse, certainunicellular, eukaryotic chlorella-like green algae. Accordingly, anysusceptible microalgae can be lysed by infecting the culture with asuitable chlorella virus. Methods of infecting species of Chlorella witha chlorella virus are known. See for example Adv. Virus Res. 2006;66:293-336; Virology, 1999 Apr. 25; 257(1):15-23; Virology, 2004 Jan. 5;318(1):214-23; Nucleic Acids Symp. Ser. 2000; (44):161-2; J. Virol. 2006March; 80(5):2437-44; and Annu. Rev. Microbiol. 1999; 53:447-94.

In another embodiment of the present invention, the step of lysing amicroorganism comprises autolysis. In this embodiment, a microorganismaccording to the invention is genetically engineered to produce a lyticprotein that will lyse the microorganism. This lytic gene can beexpressed using an inducible promoter so that the cells can first begrown to a desirable density in a fermentor, followed by induction ofthe promoter to express the lytic gene to lyse the cells. In oneembodiment, the lytic gene encodes a polysaccharide-degrading enzyme. Incertain other embodiments, the lytic gene is a gene from a lytic virus.Thus, for example, a lytic gene from a Chlorella virus can be expressedin an algal cell; see Virology 260, 308-315 (1999); FEMS MicrobiologyLetters 180 (1999) 45-53; Virology 263, 376-387 (1999); and Virology230, 361-368 (1997). Expression of lytic genes is preferably done usingan inducible promoter, such as a promoter active in microalgae that isinduced by a stimulus such as the presence of a small molecule, light,heat, and other stimuli.

Various methods are available for separating lipids from cellularlysates produced by the above methods. For example, lipids and lipidderivatives such as fatty aldehydes, fatty alcohols, and hydrocarbonssuch as alkanes can be extracted with a hydrophobic solvent such ashexane (see Frenz et al. 1989, Enzyme Microb. Technol., 11:717). Lipidsand lipid derivatives can also be extracted using liquefaction (see forexample Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and Inoueet al. 1993, Biomass Bioenergy 6(4):269-274); oil liquefaction (see forexample Minowa et al. 1995, Fuel 74(12):1735-1738); and supercriticalCO₂ extraction (see for example Mendes et al. 2003, Inorganica ChimicaActa 356:328-334). Miao and Wu describe a protocol of the recovery ofmicroalgal lipid from a culture of Chlorella prototheocoides in whichthe cells were harvested by centrifugation, washed with distilled waterand dried by freeze drying. The resulting cell powder was pulverized ina mortar and then extracted with n-hexane. Miao and Wu, BiosourceTechnology (2006) 97:841-846.

Thus, lipids, lipid derivatives and hydrocarbons generated by themicroorganisms of the present invention can be recovered by extractionwith an organic solvent. In some cases, the preferred organic solvent ishexane. Typically, the organic solvent is added directly to the lysatewithout prior separation of the lysate components. In one embodiment,the lysate generated by one or more of the methods described above iscontacted with an organic solvent for a period of time sufficient toallow the lipid and/or hydrocarbon components to form a solution withthe organic solvent. In some cases, the solution can then be furtherrefined to recover specific desired lipid or hydrocarbon components.Hexane extraction methods are well known in the art.

Lipids and lipid derivatives such as fatty aldehydes, fatty alcohols,and hydrocarbons such as alkanes produced by cells as described hereincan be modified by the use of one or more enzymes, including a lipase,as described above. When the hydrocarbons are in the extracellularenvironment of the cells, the one or more enzymes can be added to thatenvironment under conditions in which the enzyme modifies thehydrocarbon or completes its synthesis from a hydrocarbon precursor.Alternatively, the hydrocarbons can be partially, or completely,isolated from the cellular material before addition of one or morecatalysts such as enzymes. Such catalysts are exogenously added, andtheir activity occurs outside the cell or in vitro.

Thus, lipids and hydrocarbons produced by cells in vivo, orenzymatically modified in vitro, as described herein can be optionallyfurther processed by conventional means. The processing can include“cracking” to reduce the size, and thus increase the hydrogen:carbonratio, of hydrocarbon molecules. Catalytic and thermal cracking methodsare routinely used in hydrocarbon and triglyceride oil processing.Catalytic methods involve the use of a catalyst, such as a solid acidcatalyst. The catalyst can be silica-alumina or a zeolite, which resultin the heterolytic, or asymmetric, breakage of a carbon-carbon bond toresult in a carbocation and a hydride anion. These reactiveintermediates then undergo either rearrangement or hydride transfer withanother hydrocarbon. The reactions can thus regenerate the intermediatesto result in a self-propagating chain mechanism. Hydrocarbons can alsobe processed to reduce, optionally to zero, the number of carbon-carbondouble, or triple, bonds therein. Hydrocarbons can also be processed toremove or eliminate a ring or cyclic structure therein. Hydrocarbons canalso be processed to increase the hydrogen:carbon ratio. This caninclude the addition of hydrogen (“hydrogenation”) and/or the “cracking”of hydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described in SectionIV herein.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manufacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is an urgent need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass or otherwise that meets the appropriateASTM specification can be defined as diesel fuel (ASTM D975), jet fuel(ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTMD6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

Biodiesel is a liquid which varies in color—between golden and darkbrown-depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear. Typically, biodiesel comprises C14-C18 alkyl esters.Various processes convert biomass or a lipid produced and isolated asdescribed herein to diesel fuels. A preferred method to producebiodiesel is by transesterification of a lipid as described herein. Apreferred alkyl ester for use as biodiesel is a methyl ester or ethylester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B100.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers. Existing oil boilers may contain rubber parts and may requireconversion to run on biodiesel. The conversion process is usuallyrelatively simple, involving the exchange of rubber parts for syntheticparts due to biodiesel being a strong solvent. Due to its strong solventpower, burning biodiesel will increase the efficiency of boilers.Biodiesel can be used as an additive in formulations of diesel toincrease the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel,which is advantageous because it has virtually no sulfur content.Biodiesel is a better solvent than petrodiesel and can be used to breakdown deposits of residues in the fuel lines of vehicles that havepreviously been run on petrodiesel.

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel.

The lipid profile of the biodiesel is usually highly similar to thelipid profile of the feedstock oil. Other oils provided by the methodsand compositions of the invention can be subjected totransesterification to yield biodiesel with lipid profiles including (a)at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% C10; (d) atleast 2% C12; and (3) at least 30% C8-C14.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases. In a base-catalyzed transesterification process, thetriacylglycerides are reacted with an alcohol, such as methanol orethanol, in the presence of an alkaline catalyst, typically potassiumhydroxide. This reaction forms methyl or ethyl esters and glycerin(glycerol) as a byproduct.

Animal and plant oils are typically made of triglycerides which areesters of free fatty acids with the trihydric alcohol, glycerol. Intransesterification, the glycerol in a triacylglyceride (TAG) isreplaced with a short-chain alcohol such as methanol or ethanol. Atypical reaction scheme is as follows:

In this reaction, the alcohol is deprotonated with a base to make it astronger nucleophile. Commonly, ethanol or methanol is used in vastexcess (up to 50-fold). Normally, this reaction will proceed eitherexceedingly slowly or not at all. Heat, as well as an acid or base canbe used to help the reaction proceed more quickly. The acid or base arenot consumed by the transesterification reaction, thus they are notreactants but catalysts. Almost all biodiesel has been produced usingthe base-catalyzed technique as it requires only low temperatures andpressures and produces over 98% conversion yield (provided the startingoil is low in moisture and free fatty acids).

Transesterification has also been carried out, as discussed above, usingan enzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80° C., and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1. Lipasessuitable for use in transesterification include, but are not limited to,those listed in Table 7. Other examples of lipases useful fortransesterification are found in, e.g. U.S. Pat. Nos. 4,798,793;4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipasesinclude, but are not limited to, lipases produced by microorganisms ofRhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida,and Humicola and pancreas lipase.

TABLE 7 Lipases suitable for use in transesterification. Aspergillusniger lipase ABG73614, Candida antarctica lipase B (novozym-435)CAA83122, Candida cylindracea lipase AAR24090, Candida lipolytica lipase(Lipase L; Amano Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g.,Lipase-OF; Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase(Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopusoryzae lipase (Lipase F) AAF32408, Serratia marcescens lipase (SMEnzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P(Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd.,Nagoya, Japan)

One challenge to using a lipase for the production of fatty acid esterssuitable for biodiesel is that the price of lipase is much higher thanthe price of sodium hydroxide (NaOH) used by the strong base process.This challenge has been addressed by using an immobilized lipase, whichcan be recycled. However, the activity of the immobilized lipase must bemaintained after being recycled for a minimum number of cycles to allowa lipase-based process to compete with the strong base process in termsof the production cost. Immobilized lipases are subject to poisoning bythe lower alcohols typically used in transesterification. U.S. Pat. No.6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods forenhancing the activity of immobilized lipases and regeneratingimmobilized lipases having reduced activity. Some suitable methodsinclude immersing an immobilized lipase in an alcohol having a carbonatom number not less than 3 for a period of time, preferably from 0.5-48hours, and more preferably from 0.5-1.5 hours. Some suitable methodsalso include washing a deactivated immobilized lipase with an alcoholhaving a carbon atom number not less than 3 and then immersing thedeactivated immobilized lipase in a vegetable oil for 0.5-48 hours.

In particular embodiments, a recombinant lipase is expressed in the samemicroorganisms that produce the lipid on which the lipase acts. Suitablerecombinant lipases include those listed above in Table 7 and/or havingGenBank Accession numbers listed above in Table 7, or a polypeptide thathas at least 70% amino acid identity with one of the lipases listedabove in Table 7 and that exhibits lipase activity. In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth. DNA encoding the lipase and selectablemarker is preferably codon-optimized cDNA. Methods of recoding genes forexpression in microalgae are described in U.S. Pat. No. 7,135,290.

The common international standard for biodiesel is EN 14214. ASTM D6751is the most common biodiesel standard referenced in the United Statesand Canada. Germany uses DIN EN 14214 and the UK requires compliancewith BS EN 14214. Basic industrial tests to determine whether theproducts conform to these standards typically include gaschromatography, HPLC, and others. Biodiesel meeting the qualitystandards is very non-toxic, with a toxicity rating (LD₅₀) of greaterthan 50 mL/kg.

Although biodiesel that meets the ASTM standards has to be non-toxic,there can be contaminants which tend to crystallize and/or precipitateand fall out of solution as sediment. Sediment formation is particularlya problem when biodiesel is used at lower temperatures. The sediment orprecipitates may cause problems such as decreasing fuel flow, cloggingfuel lines, clogging filters, etc. Processes are well-known in the artthat specifically deal with the removal of these contaminants andsediments in biodiesel in order to produce a higher quality product.Examples for such processes include, but are not limited to,pretreatment of the oil to remove contaiminants such as phospholipidsand free fatty acids (e.g., degumming, caustic refining and silicaadsorbant filtration) and cold filtration. Cold filtration is a processthat was developed specifically to remove any particulates and sedimentsthat are present in the biodiesel after production. This process coolsthe biodiesel and filters out any sediments or precipitates that mightform when the fuel is used at a lower temperature. Such a process iswell known in the art and is described in US Patent ApplicationPublication No. 2007-0175091. Suitable methods may include cooling thebiodiesel to a temperature of less than about 38° C. so that theimpurities and contaminants precipitate out as particulates in thebiodiesel liquid. Diatomaceous earth or other filtering material maythen added to the cooled biodiesel to form a slurry, which may thenfiltered through a pressure leaf or other type of filter to remove theparticulates. The filtered biodiesel may then be run through a polishfilter to remove any remaining sediments and diatomaceous earth, so asto produce the final biodiesel product.

Example 14 described the production of biodiesel using triglyceride oilfrom Prototheca moriformis. The Cold Soak Filterability by the ASTMD6751 A1 method of the biodiesel produced in Example 14 was 120 secondsfor a volume of 300 ml. This test involves filtration of 300 ml of B100,chilled to 40° F. for 16 hours, allowed to warm to room temp, andfiltered under vacuum using 0.7 micron glass fiber filter with stainlesssteel support. Oils of the invention can be transesterified to generatebiodiesel with a cold soak time of less than 120 seconds, less than 100seconds, and less than 90 seconds.

Subsequent processes may also be used if the biodiesel will be used inparticularly cold temperatures. Such processes include winterization andfractionation. Both processes are designed to improve the cold flow andwinter performance of the fuel by lowering the cloud point (thetemperature at which the biodiesel starts to crystallize). There areseveral approaches to winterizing biodiesel. One approach is to blendthe biodiesel with petroleum diesel. Another approach is to useadditives that can lower the cloud point of biodiesel. Another approachis to remove saturated methyl esters indiscriminately by mixing inadditives and allowing for the crystallization of saturates and thenfiltering out the crystals. Fractionation selectively separates methylesters into individual components or fractions, allowing for the removalor inclusion of specific methyl esters. Fractionation methods includeurea fractionation, solvent fractionation and thermal distillation.

Another valuable fuel provided by the methods of the present inventionis renewable diesel, which comprises alkanes, such as C10:0, C12:0,C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel.High quality renewable diesel conforms to the ASTM D975 standard. Thelipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In one embodiment, the method for producing renewable diesel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing the microorganism to produce a lysate, (c) isolatinglipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696. Some suitable methods may include mechanicalpressing and centrifuging.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet an ASTM D975standard while leaving components that are heavier than desired formeeting the D975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

In one embodiment of the method for producing renewable diesel, treatingthe lipid to produce an alkane is performed by hydrotreating of thelipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300° to 660°F., with pressure sufficient to keep the water primarily as a liquid,100 to 170 standard atmosphere (atm). Reaction times are on the order of15 to 30 minutes. After the reaction is completed, the organics areseparated from the water. Thereby a distillate suitable for diesel isproduced.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. No. 5,475,160(hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

One suitable method for the hydrogenation of triglycerides includespreparing an aqueous solution of copper, zinc, magnesium and lanthanumsalts and another solution of alkali metal or preferably, ammoniumcarbonate. The two solutions may be heated to a temperature of about 20°C. to about 85° C. and metered together into a precipitation containerat rates such that the pH in the precipitation container is maintainedbetween 5.5 and 7.5 in order to form a catalyst. Additional water may beused either initially in the precipitation container or addedconcurrently with the salt solution and precipitation solution. Theresulting precipitate may then be thoroughly washed, dried, calcined atabout 300° C. and activated in hydrogen at temperatures ranging fromabout 100° C. to about 400° C. One or more triglycerides may then becontacted and reacted with hydrogen in the presence of theabove-described catalyst in a reactor. The reactor may be a trickle bedreactor, fixed bed gas-solid reactor, packed bubble column reactor,continuously stirred tank reactor, a slurry phase reactor, or any othersuitable reactor type known in the art. The process may be carried outeither batchwise or in continuous fashion. Reaction temperatures aretypically in the range of from about 170° C. to about 250° C. whilereaction pressures are typically in the range of from about 300 psig toabout 2000 psig. Moreover, the molar ratio of hydrogen to triglyceridein the process of the present invention is typically in the range offrom about 20:1 to about 700:1. The process is typically carried out ata weight hourly space velocity (WHSV) in the range of from about 0.1hr⁻¹ to about 5 hr⁻¹. One skilled in the art will recognize that thetime period required for reaction will vary according to the temperatureused, the molar ratio of hydrogen to triglyceride, and the partialpressure of hydrogen. The products produced by the such hydrogenationprocesses include fatty alcohols, glycerol, traces of paraffins andunreacted triglycerides. These products are typically separated byconventional means such as, for example, distillation, extraction,filtration, crystallization, and the like.

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes. Solidcatalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Suitable methods for the deoxygenation of an oil includes heating an oilto a temperature in the range of from about 350° F. to about 550° F. andcontinuously contacting the heated oil with nitrogen under at leastpressure ranging from about atmospheric to above for at least about 5minutes.

Suitable methods for isomerization includes using alkali isomerizationand other oil isomerization known in the art.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the triglyceride feed. The triglyceride molecule isreduced to four hydrocarbon molecules under hydroprocessing conditions:a propane molecule and three heavier hydrocarbon molecules, typically inthe C8 to C18 range.

Thus, in one embodiment, the product of one or more chemical reaction(s)performed on lipid compositions of the invention is an alkane mixturethat comprises ASTM D975 renewable diesel. Production of hydrocarbons bymicroorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol(2005) 66: 486-496 and A Look Back at the U.S. Department of Energy'sAquatic Species Program: Biodiesel from Algae, NREL/TP-580-24190, JohnSheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).

The distillation properties of a diesel fuel is described in terms ofT10-T90 (temperature at 10% and 90%, respectively, volume distilled).Renewable diesel was produced from Prototheca moriformis triglycerideoil and is described in Example 14. The T10-T90 of the material producedin Example 14 was 57.9° C. Methods of hydrotreating, isomerization, andother covalent modification of oils disclosed herein, as well as methodsof distillation and fractionation (such as cold filtration) disclosedherein, can be employed to generate renewable diesel compositions withother T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65° C.using triglyceride oils produced according to the methods disclosedherein.

The T10 of the material produced in Example 14 was 242.1° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10 values, such as T10 between180 and 295, between 190 and 270, between 210 and 250, between 225 and245, and at least 290.

The T90 of the material produced in Example 14 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein can be employed to generaterenewable diesel compositions with other T90 values, such as T90 between280 and 380, between 290 and 360, between 300 and 350, between 310 and340, and at least 290.

The FBP of the material produced in Example 14 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other FBP values, such as FBP between290 and 400, between 300 and 385, between 310 and 370, between 315 and360, and at least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% C10; (d) atleast 2% C12; and (3) at least 30% C8-C14.

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

The present invention also provides methods to produce jet fuel. Jetfuel is clear to straw colored. The most common fuel is anunleaded/paraffin oil-based fuel classified as Aeroplane A-1, which isproduced to an internationally standardized set of specifications. Jetfuel is a mixture of a large number of different hydrocarbons, possiblyas many as a thousand or more. The range of their sizes (molecularweights or carbon numbers) is restricted by the requirements for theproduct, for example, freezing point or smoke point. Kerosone-typeAeroplane fuel (including Jet A and Jet A-1) has a carbon numberdistribution between about 8 and 16 carbon numbers. Wide-cut ornaphta-type Aeroplane fuel (including Jet B) typically has a carbonnumber distribution between about 5 and 15 carbons.

Both Aeroplanes (Jet A and Jet B) may contain a number of additives.Useful additives include, but are not limited to, antioxidants,antistatic agents, corrosion inhibitors, and fuel system icing inhibitor(FSII) agents. Antioxidants prevent gumming and usually, are based onalkylated phenols, for example, AO-30, AO-31, or AO-37. Antistaticagents dissipate static electricity and prevent sparking. Stadis 450with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, isan example. Corrosion inhibitors, e.g., DCI-4A is used for civilian andmilitary fuels and DCI-6A is used for military fuels. FSII agents,include, e.g., Di-EGME.

In one embodiment of the invention, a jet fuel is produced by blendingalgal fuels with existing jet fuel. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. The lipidsproduced by the method of the present invention can be converted toolefins. The process involves flowing the lipids produced through an FCCzone and collecting a product stream comprised of olefins, which isuseful as a jet fuel. The lipids produced are contacted with a crackingcatalyst at cracking conditions to provide a product stream comprisingolefins and hydrocarbons useful as jet fuel.

In one embodiment, the method for producing jet fuel comprises (a)cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced. In one embodiment of themethod for producing a jet fuel, the lipid composition can be flowedthrough a fluid catalytic cracking zone, which, in one embodiment, maycomprise contacting the lipid composition with a cracking catalyst atcracking conditions to provide a product stream comprising C₂-C₅olefins.

In certain embodiments of this method, it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C₂-C₅ olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

Suitable FCC catalysts generally comprise at least two components thatmay or may not be on the same matrix. In some embodiments, both twocomponents may be circulated throughout the entire reaction vessel. Thefirst component generally includes any of the well-known catalysts thatare used in the art of fluidized catalytic cracking, such as an activeamorphous clay-type catalyst and/or a high activity, crystallinemolecular sieve. Molecular sieve catalysts may be preferred overamorphous catalysts because of their much-improved selectivity todesired products. IN some preferred embodiments, zeolites may be used asthe molecular sieve in the FCC processes. Preferably, the first catalystcomponent comprises a large pore zeolite, such as an Y-type zeolite, anactive alumina material, a binder material, comprising either silica oralumina and an inert filler such as kaolin.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149° C. to about 316° C. (300° F.to 600° F.). The catalyst is flowed from a blending vessel to the riserwhere it contacts the lipid composition for a time of abort 2 seconds orless.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst to lipidcomposition ratio which can vary from about 5 to about 20 and preferablyfrom about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700° C. (1292° F.) to about 760° C. (1400° F.) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO). HDO means removal of oxygen bymeans of hydrogen, that is, oxygen is removed while breaking thestructure of the material. Olefinic double bonds are hydrogenated andany sulphur and nitrogen compounds are removed. Sulphur removal iscalled hydrodesulphurization (HDS). Pretreatment and purity of the rawmaterials (lipid composition or the lipids) contribute to the servicelife of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerzation reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulphur compounds, andlight hydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerzation is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step, i.e. HDO step, of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle. In the HDO step, thepressure varies between 20 and 150 bar, preferably between 50 and 100bar, and the temperature varies between 200 and 500° C., preferably inthe range of 300-400° C. In the HDO step, known hydrogenation catalystscontaining metals from Group VII and/or VIB of the Periodic System maybe used. Preferably, the hydrogenation catalysts are supported Pd, Pt,Ni, NiMo or a CoMo catalysts, the support being alumina and/or silica.Typically, NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50 400° C. and at hydrogen pressures of 1 200 bar,preferably at a temperature between 150 and 250° C. and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapour or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner. In theisomerzation step, the pressure varies in the range of 20 150 bar,preferably in the range of 20 100 bar, the temperature being between 200and 500° C., preferably between 300 and 400° C. In the isomerizationstep, isomerization catalysts known in the art may be used. Suitableisomerization catalysts contain molecular sieve and/or a metal fromGroup VII and/or a carrier. Preferably, the isomerization catalystcontains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pdor Ni and Al₂O₃ or SiO₂. Typical isomerization catalysts are, forexample, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂. The isomerization step and the HDO step may be carriedout in the same pressure vessel or in separate pressure vessels.Optional prehydrogenation may be carried out in a separate pressurevessel or in the same pressure vessel as the HDO and isomerizationsteps.

Thus, in one embodiment, the product of the one or more chemicalreactions is an alkane mixture that comprises ASTM D1655 jet fuel. Insome embodiments, the composition comforming to the specification ofASTM 1655 jet fuel has a sulfur content that is less than 10 ppm. Inother embodiments, the composition conforming to the specification ofASTM 1655 jet fuel has a T10 value of the distillation curve of lessthan 205° C. In another embodiment, the composition conforming to thespecification of ASTM 1655 jet fuel has a final boiling point (FBP) ofless than 300° C. In another embodiment, the composition conforming tothe specification of ASTM 1655 jet fuel has a flash point of at least38° C. In another embodiment, the composition conforming to thespecification of ASTM 1655 jet fuel has a density between 775K/M³ and840K/M³. In yet another embodiment, the composition conforming to thespecification of ASTM 1655 jet fuel has a freezing point that is below−47° C. In another embodiment, the composition conforming to thespecification of ASTM 1655 jet fuel has a net Heat of Combustion that isat least 42.8 MJ/K. In another embodiment, the composition conforming tothe specification of ASTM 1655 jet fuel has a hydrogen content that isat least 13.4 mass %. In another embodiment, the composition conformingto the specification of ASTM 1655 jet fuel has a thermal stability, astested by quantitative gravimetric JFTOT at 260° C., that is below 3 mmof Hg. In another embodiment, the composition conforming to thespecification of ASTM 1655 jet fuel has an existent gum that is below 7mg/dl.

Thus, the present invention discloses a variety of methods in whichchemical modification of microalgal lipid is undertaken to yieldproducts useful in a variety of industrial and other applications.Examples of processes for modifying oil produced by the methodsdisclosed herein include, but are not limited to, hydrolysis of the oil,hydroprocessing of the oil, and esterification of the oil. Themodification of the microalgal oil produces basic oleochemicals that canbe further modified into selected derivative oleochemicals for a desiredfunction. In a manner similar to that described above with reference tofuel producing processes, these chemical modifications can also beperformed on oils generated from the microbial cultures describedherein. Examples of basic oleochemicals include, but are not limited to,soaps, fatty acids, fatty acid methyl esters, and glycerol. Examples ofderivative oleochemicals include, but are not limited to, fattynitriles, esters, dimer acids, quats, surfactants, fatty alkanolamides,fatty alcohol sulfates, resins, emulsifiers, fatty alcohols, olefins,and higher alkanes.

Hydrolysis of the fatty acid constituents from the glycerolipidsproduced by the methods of the invention yields free fatty acids thatcan be derivatized to produce other useful chemicals. Hydrolysis occursin the presence of water and a catalyst which may be either an acid or abase. The liberated free fatty acids can be derivatized to yield avariety of products, as reported in the following: U.S. Pat. No.5,304,664 (Highly sulfated fatty acids); U.S. Pat. No. 7,262,158(Cleansing compositions); U.S. Pat. No. 7,115,173 (Fabric softenercompositions); U.S. Pat. No. 6,342,208 (Emulsions for treating skin);U.S. Pat. No. 7,264,886 (Water repellant compositions); U.S. Pat. No.6,924,333 (Paint additives); U.S. Pat. No. 6,596,768 (Lipid-enrichedruminant feedstock); and U.S. Pat. No. 6,380,410 (Surfactants fordetergents and cleaners).

With regard to hydrolysis, in one embodiment of the invention, atriglyceride oil is optionally first hydrolyzed in a liquid medium suchas water or sodium hydroxide so as to obtain glycerol and soaps. Thereare various suitable triglyceride hydrolysis methods, including, but notlimited to, saponification, acid hydrolysis, alkaline hydrolysis,enzymatic hydrolysis (referred herein as splitting), and hydrolysisusing hot-compressed water. One skilled in the art will recognize that atriglyceride oil need not be hydrolyzed in order to produce anoleochemical; rather, the oil may be converted directly to the desiredoleochemical by other known process. For example, the triglyceride oilmay be directly converted to a methyl ester fatty acid throughesterification.

In some embodiments, catalytic hydrolysis of the oil produced by methodsdisclosed herein occurs by splitting the oil into glycerol and fattyacids. As discussed above, the fatty acids may then be further processedthrough several other modifications to obtained derivativeoleochemicals. For example, in one embodiment the fatty acids mayundergo an amination reaction to produce fatty nitrogen compounds. Inanother embodiment, the fatty acids may undergo ozonolysis to producemono- and dibasic-acids.

In other embodiments hydrolysis may occur via the, splitting of oilsproduced herein to create oleochemicals. In some preferred embodimentsof the invention, a triglyceride oil may be split before other processesis performed. One skilled in the art will recognize that there are manysuitable triglyceride splitting methods, including, but not limited to,enzymatic splitting and pressure splitting.

Generally, enzymatic oil splitting methods use enzymes, lipases, asbiocatalysts acting on a water/oil mixture. Enzymatic splitting thensplits the oil or fat, respectively, is into glycerol and free fattyacids. The glycerol may then migrates into the water phase whereas theorganic phase enriches with free fatty acids.

The enzymatic splitting reactions generally take place at the phaseboundary between organic and aqueous phase, where the enzyme is presentonly at the phase boundary. Triglycerides that meet the phase boundarythen contribute to or participate in the splitting reaction. As thereaction proceeds, the occupation density or concentration of fattyacids still chemically bonded as glycerides, in comparison to free fattyacids, decreases at the phase boundary so that the reaction is sloweddown. In certain embodiments, enzymatic splitting may occur at roomtemperature. One of ordinary skill in the art would know the suitableconditions for splitting oil into the desired fatty acids.

By way of example, the reaction speed can be accelerated by increasingthe interface boundary surface. Once the reaction is complete, freefatty acids are then separated from the organic phase freed from enzyme,and the residue which still contains fatty acids chemically bonded asglycerides is fed back or recycled and mixed with fresh oil or fat to besubjected to splitting. In this manner, recycled glycerides are thensubjected to a further enzymatic splitting process. In some embodiments,the free fatty acids are extracted from an oil or fat partially split insuch a manner. In that way, if the chemically bound fatty acids(triglycerides) are returned or fed back into the splitting process, theenzyme consumption can be drastically reduced.

The splitting degree is determined as the ratio of the measured acidvalue divided by the theoretically possible acid value which can becomputed for a given oil or fat. Preferably, the acid value is measuredby means of titration according to standard common methods.Alternatively, the density of the aqueous glycerol phase can be taken asa measure for the splitting degree.

In one embodiment, the slitting process as described herein is alsosuitable for splitting the mono-, di- and triglyceride that arecontained in the so-called soap-stock from the alkali refining processesof the produced oils. In this manner, the soap-stock can bequantitatively converted without prior saponification of the neutraloils into the fatty acids. For this purpose, the fatty acids beingchemically bonded in the soaps are released, preferably beforesplitting, through an addition of acid. In certain embodiments, a buffersolution is used in addition to water and enzyme for the splittingprocess.

In one embodiment, oils produced in accordance with the methods of theinvention can also be subjected to saponification as a method ofhydrolysis Animal and plant oils are typically made of triacylglycerols(TAGs), which are esters of fatty acids with the trihydric alcohol,glycerol. In an alkaline hydrolysis reaction, the glycerol in a TAG isremoved, leaving three carboxylic acid anions that can associate withalkali metal cations such as sodium or potassium to produce fatty acidsalts. In this scheme, the carboxylic acid constituents are cleaved fromthe glycerol moiety and replaced with hydroxyl groups. The quantity ofbase (e.g., KOH) that is used in the reaction is determined by thedesired degree of saponification. If the objective is, for example, toproduce a soap product that comprises some of the oils originallypresent in the TAG composition, an amount of base insufficient toconvert all of the TAGs to fatty acid salts is introduced into thereaction mixture. Normally, this reaction is performed in an aqueoussolution and proceeds slowly, but may be expedited by the addition ofheat. Precipitation of the fatty acid salts can be facilitated byaddition of salts, such as water-soluble alkali metal halides (e.g.,NaCl or KCl), to the reaction mixture. Preferably, the base is an alkalimetal hydroxide, such as NaOH or KOH. Alternatively, other bases, suchas alkanolamines, including for example triethanolamine andaminomethylpropanol, can be used in the reaction scheme. In some cases,these alternatives may be preferred to produce a clear soap product.

In some methods, the first step of chemical modification may behydroprocessing to saturate double bonds, followed by deoxygenation atelevated temperature in the presence of hydrogen and a catalyst. Inother methods, hydrogenation and deoxygenation may occur in the samereaction. In still other methods deoxygenation occurs beforehydrogenation. Isomerization may then be optionally performed, also inthe presence of hydrogen and a catalyst. Finally, gases and naphthacomponents can be removed if desired. For example, see U.S. Pat. No.5,475,160 (hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

In some embodiments of the invention, the triglyceride oils arepartially or completely deoxygenated. The deoxygenation reactions formdesired products, including, but not limited to, fatty acids, fattyalcohols, polyols, ketones, and aldehydes. In general, without beinglimited by any particular theory, the deoxygenation reactions involve acombination of various different reaction pathways, including withoutlimitation: hydrogenolysis, hydrogenation, consecutivehydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation,and combined hydrogenation-hydrogenolysis reactions, resulting in atleast the partial removal of oxygen from the fatty acid or fatty acidester to produce reaction products, such as fatty alcohols, that can beeasily converted to the desired chemicals by further processing. Forexample, in one embodiment, a fatty alcohol may be converted to olefinsthrough FCC reaction or to higher alkanes through a condensationreaction.

One such chemical modification is hydrogenation, which is the additionof hydrogen to double bonds in the fatty acid constituents ofglycerolipids or of free fatty acids. The hydrogenation process permitsthe transformation of liquid oils into semi-solid or solid fats, whichmay be more suitable for specific applications.

Hydrogenation of oil produced by the methods described herein can beperformed in conjunction with one or more of the methods and/ormaterials provided herein, as reported in the following: U.S. Pat. No.7,288,278 (Food additives or medicaments); U.S. Pat. No. 5,346,724(Lubrication products); U.S. Pat. No. 5,475,160 (Fatty alcohols); U.S.Pat. No. 5,091,116 (Edible oils); U.S. Pat. No. 6,808,737 (Structuralfats for margarine and spreads); U.S. Pat. No. 5,298,637(Reduced-calorie fat substitutes); U.S. Pat. No. 6,391,815(Hydrogenation catalyst and sulfur adsorbent); U.S. Pat. No. 5,233,099and U.S. Pat. No. 5,233,100 (Fatty alcohols); U.S. Pat. No. 4,584,139(Hydrogenation catalysts); U.S. Pat. No. 6,057,375 (Foam suppressingagents); and U.S. Pat. No. 7,118,773 (Edible emulsion spreads).

One skilled in the art will recognize that various processes may be usedto hydrogenate carbohydrates. One suitable method includes contactingthe carbohydrate with hydrogen or hydrogen mixed with a suitable gas anda catalyst under conditions sufficient in a hydrogenation reactor toform a hydrogenated product. The hydrogenation catalyst generally caninclude Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or anycombination thereof, either alone or with promoters such as W, Mo, Au,Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof.Other effective hydrogenation catalyst materials include eithersupported nickel or ruthenium modified with rhenium. In an embodiment,the hydrogenation catalyst also includes any one of the supports,depending on the desired functionality of the catalyst. Thehydrogenation catalysts may be prepared by methods known to those ofordinary skill in the art.

In some embodiments the hydrogenation catalyst includes a supportedGroup VIII metal catalyst and a metal sponge material (e.g., a spongenickel catalyst). Raney nickel provides an example of an activatedsponge nickel catalyst suitable for use in this invention. In otherembodiment, the hydrogenation reaction in the invention is performedusing a catalyst comprising a nickel-rhenium catalyst or atungsten-modified nickel catalyst. One example of a suitable catalystfor the hydrogenation reaction of the invention is a carbon-supportednickel-rhenium catalyst.

In an embodiment, a suitable Raney nickel catalyst may be prepared bytreating an alloy of approximately equal amounts by weight of nickel andaluminum with an aqueous alkali solution, e.g., containing about 25weight % of sodium hydroxide. The aluminum is selectively dissolved bythe aqueous alkali solution resulting in a sponge shaped materialcomprising mostly nickel with minor amounts of aluminum. The initialalloy includes promoter metals (i.e., molybdenum or chromium) in theamount such that about 1 to 2 weight % remains in the formed spongenickel catalyst. In another embodiment, the hydrogenation catalyst isprepared using a solution of ruthenium(III) nitrosylnitrate, ruthenium(III) chloride in water to impregnate a suitable support material. Thesolution is then dried to form a solid having a water content of lessthan about 1% by weight. The solid may then be reduced at atmosphericpressure in a hydrogen stream at 300° C. (uncalcined) or 400° C.(calcined) in a rotary ball furnace for 4 hours. After cooling andrendering the catalyst inert with nitrogen, 5% by volume of oxygen innitrogen is passed over the catalyst for 2 hours.

In certain embodiments, the catalyst described includes a catalystsupport. The catalyst support stabilizes and supports the catalyst. Thetype of catalyst support used depends on the chosen catalyst and thereaction conditions. Suitable supports for the invention include, butare not limited to, carbon, silica, silica-alumina, zirconia, titania,ceria, vanadia, nitride, boron nitride, heteropolyacids, hydroxyapatite,zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene andany combination thereof.

The catalysts used in this invention can be prepared using conventionalmethods known to those in the art. Suitable methods may include, but arenot limited to, incipient wetting, evaporative impregnation, chemicalvapor deposition, wash-coating, magnetron sputtering techniques, and thelike.

The conditions for which to carry out the hydrogenation reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate reaction conditions. In general, thehydrogenation reaction is conducted at temperatures of 80° C. to 250°C., and preferably at 90° C. to 200° C., and most preferably at 100° C.to 150° C. In some embodiments, the hydrogenation reaction is conductedat pressures from 500 KPa to 14000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention may include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof. As used herein, theterm “external hydrogen” refers to hydrogen that does not originate fromthe biomass reaction itself, but rather is added to the system fromanother source.

In some embodiments of the invention, it is desirable to convert thestarting carbohydrate to a smaller molecule that will be more readilyconverted to desired higher hydrocarbons. One suitable method for thisconversion is through a hydrogenolysis reaction. Various processes areknown for performing hydrogenolysis of carbohydrates. One suitablemethod includes contacting a carbohydrate with hydrogen or hydrogenmixed with a suitable gas and a hydrogenolysis catalyst in ahydrogenolysis reactor under conditions sufficient to form a reactionproduct comprising smaller molecules or polyols. As used herein, theterm “smaller molecules or polyols” includes any molecule that has asmaller molecular weight, which can include a smaller number of carbonatoms or oxygen atoms than the starting carbohydrate. In an embodiment,the reaction products include smaller molecules that include polyols andalcohols. Someone of ordinary skill in the art would be able to choosethe appropriate method by which to carry out the hydrogenolysisreaction.

In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may beconverted to propylene glycol, ethylene glycol, and glycerol using ahydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo,W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or anycombination thereof, either alone or with promoters such as Au, Ag, Cr,Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. Thehydrogenolysis catalyst may also include a carbonaceous pyropolymercatalyst containing transition metals (e.g., chromium, molybdemum,tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals(e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium,iridium, and osmium). In certain embodiments, the hydrogenolysiscatalyst may include any of the above metals combined with an alkalineearth metal oxide or adhered to a catalytically active support. Incertain embodiments, the catalyst described in the hydrogenolysisreaction may include a catalyst support as described above for thehydrogenation reaction.

The conditions for which to carry out the hydrogenolysis reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate conditions to use to carry out thereaction. In general, they hydrogenolysis reaction is conducted attemperatures of 110° C. to 300° C., and preferably at 170° C. to 220°C., and most preferably at 200° C. to 225° C. In some embodiments, thehydrogenolysis reaction is conducted under basic conditions, preferablyat a pH of 8 to 13, and even more preferably at a pH of 10 to 12. Insome embodiments, the hydrogenolysis reaction is conducted at pressuresin a range between 60 KPa and 16500 KPa, and preferably in a rangebetween 1700 KPa and 14000 KPa, and even more preferably between 4800KPa and 11000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention can include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof.

In some embodiments, the reaction products discussed above may beconverted into higher hydrocarbons through a condensation reaction in acondensation reactor (shown schematically as condensation reactor 110 inFIG. 1). In such embodiments, condensation of the reaction productsoccurs in the presence of a catalyst capable of forming higherhydrocarbons. While not intending to be limited by theory, it isbelieved that the production of higher hydrocarbons proceeds through astepwise addition reaction including the formation of carbon-carbon, orcarbon-oxygen bond. The resulting reaction products include any numberof compounds containing these moieties, as described in more detailbelow.

In certain embodiments, suitable condensation catalysts include an acidcatalyst, a base catalyst, or an acid/base catalyst. As used herein, theterm “acid/base catalyst” refers to a catalyst that has both an acid anda base functionality. In some embodiments the condensation catalyst caninclude, without limitation, zeolites, carbides, nitrides, zirconia,alumina, silica, aluminosilicates, phosphates, titanium oxides, zincoxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandiumoxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides,hydroxides, heteropolyacids, inorganic acids, acid modified resins, basemodified resins, and any combination thereof. In some embodiments, thecondensation catalyst can also include a modifier. Suitable modifiersinclude La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and anycombination thereof. In some embodiments, the condensation catalyst canalso include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe,Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys,and any combination thereof.

In certain embodiments, the catalyst described in the condensationreaction may include a catalyst support as described above for thehydrogenation reaction. In certain embodiments, the condensationcatalyst is self-supporting. As used herein, the term “self-supporting”means that the catalyst does not need another material to serve assupport. In other embodiments, the condensation catalyst in used inconjunction with a separate support suitable for suspending thecatalyst. In an embodiment, the condensation catalyst support is silica.

The conditions under which the condensation reaction occurs will varybased on the type of starting material and the desired products. One ofordinary skill in the art, with the benefit of this disclosure, willrecognize the appropriate conditions to use to carry out the reaction.In some embodiments, the condensation reaction is carried out at atemperature at which the thermodynamics for the proposed reaction arefavorable. The temperature for the condensation reaction will varydepending on the specific starting polyol or alcohol. In someembodiments, the temperature for the condensation reaction is in a rangefrom 80° C. to 500° C., and preferably from 125° C. to 450° C., and mostpreferably from 125° C. to 250° C. In some embodiments, the condensationreaction is conducted at pressures in a range between 0 Kpa to 9000 KPa,and preferably in a range between 0 KPa and 7000 KPa, and even morepreferably between 0 KPa and 5000 KPa.

The higher alkanes formed by the invention include, but are not limitedto, branched or straight chain alkanes that have from 4 to 30 carbonatoms, branched or straight chain alkenes that have from 4 to 30 carbonatoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenesthat have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, andketones. Suitable alkanes include, but are not limited to, butane,pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane,3-methylpentane, 2,2,-dimethylbutane, 2,3-dimethylbutane, heptane,heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane,2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane,decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene,tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane,nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane,doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, andisomers thereof. Some of these products may be suitable for use asfuels.

In some embodiments, the cycloalkanes and the cycloalkenes areunsubstituted. In other embodiments, the cycloalkanes and cycloalkenesare mono-substituted. In still other embodiments, the cycloalkanes andcycloalkenes are multi-substituted. In the embodiments comprising thesubstituted cycloalkanes and cycloalkenes, the substituted groupincludes, without limitation, a branched or straight chain alkyl having1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to12 carbon atoms, a phenyl, and any combination thereof. Suitablecycloalkanes and cycloalkenes include, but are not limited to,cyclopentane, cyclopentene, cyclohexane, cyclohexene,methyl-cyclopentane, methyl-cyclopentene, ethyl-cyclopentane,ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers andany combination thereof.

In some embodiments, the aryls formed are unsubstituted. In anotherembodiment, the aryls formed are mono-substituted. In the embodimentscomprising the substituted aryls, the substituted group includes,without limitation, a branched or straight chain alkyl having 1 to 12carbon atoms, a branched or straight chain alkylene having 1 to 12carbon atoms, a phenyl, and any combination thereof. Suitable aryls forthe invention include, but are not limited to, benzene, toluene, xylene,ethyl benzene, para xylene, meta xylene, and any combination thereof.

The alcohols produced in the invention have from 4 to 30 carbon atoms.In some embodiments, the alcohols are cyclic. In other embodiments, thealcohols are branched. In another embodiment, the alcohols are straightchained. Suitable alcohols for the invention include, but are notlimited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol,decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol,hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol,uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomersthereof.

The ketones produced in the invention have from 4 to 30 carbon atoms. Inan embodiment, the ketones are cyclic. In another embodiment, theketones are branched. In another embodiment, the ketones are straightchained. Suitable ketones for the invention include, but are not limitedto, butanone, pentanone, hexanone, heptanone, octanone, nonanone,decanone, undecanone, dodecanone, tridecanone, tetradecanone,pentadecanone, hexadecanone, heptyldecanone, octyldecanone,nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone,tetraeicosanone, and isomers thereof.

Another such chemical modification is interesterification. Naturallyproduced glycerolipids do not have a uniform distribution of fatty acidconstituents. In the context of oils, interesterification refers to theexchange of acyl radicals between two esters of different glycerolipids.The interesterification process provides a mechanism by which the fattyacid constituents of a mixture of glycerolipids can be rearranged tomodify the distribution pattern. Interesterification is a well-knownchemical process, and generally comprises heating (to about 200° C.) amixture of oils for a period (e.g., 30 minutes) in the presence of acatalyst, such as an alkali metal or alkali metal alkylate (e.g., sodiummethoxide). This process can be used to randomize the distributionpattern of the fatty acid constituents of an oil mixture, or can bedirected to produce a desired distribution pattern. This method ofchemical modification of lipids can be performed on materials providedherein, such as microbial biomass with a percentage of dry cell weightas lipid at least 20%.

Directed interesterification, in which a specific distribution patternof fatty acids is sought, can be performed by maintaining the oilmixture at a temperature below the melting point of some TAGs whichmight occur. This results in selective crystallization of these TAGs,which effectively removes them from the reaction mixture as theycrystallize. The process can be continued until most of the fatty acidsin the oil have precipitated, for example. A directedinteresterification process can be used, for example, to produce aproduct with a lower calorie content via the substitution oflonger-chain fatty acids with shorter-chain counterparts. Directedinteresterification can also be used to produce a product with a mixtureof fats that can provide desired melting characteristics and structuralfeatures sought in food additives or products (e.g., margarine) withoutresorting to hydrogenation, which can produce unwanted trans isomers.

Interesterification of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,080,853 (Nondigestible fat substitutes); U.S. Pat. No.4,288,378 (Peanut butter stabilizer); U.S. Pat. No. 5,391,383 (Ediblespray oil); U.S. Pat. No. 6,022,577 (Edible fats for food products);U.S. Pat. No. 5,434,278 (Edible fats for food products); U.S. Pat. No.5,268,192 (Low calorie nut products); U.S. Pat. No. 5,258,197 (Reducecalorie edible compositions); U.S. Pat. No. 4,335,156 (Edible fatproduct); U.S. Pat. No. 7,288,278 (Food additives or medicaments); U.S.Pat. No. 7,115,760 (Fractionation process); U.S. Pat. No. 6,808,737(Structural fats); U.S. Pat. No. 5,888,947 (Engine lubricants); U.S.Pat. No. 5,686,131 (Edible oil mixtures); and U.S. Pat. No. 4,603,188(Curable urethane compositions).

In one embodiment in accordance with the invention, transesterificationof the oil, as described above, is followed by reaction of thetransesterified product with polyol, as reported in U.S. Pat. No.6,465,642, to produce polyol fatty acid polyesters. Such anesterification and separation process may comprise the steps as follows:reacting a lower alkyl ester with polyol in the presence of soap;removing residual soap from the product mixture; water-washing anddrying the product mixture to remove impurities; bleaching the productmixture for refinement; separating at least a portion of the unreactedlower alkyl ester from the polyol fatty acid polyester in the productmixture; and recycling the separated unreacted lower alkyl ester.

Transesterification can also be performed on microbial biomass withshort chain fatty acid esters, as reported in U.S. Pat. No. 6,278,006.In general, transesterification may be performed by adding a short chainfatty acid ester to an oil in the presence of a suitable catalyst andheating the mixture. In some embodiments, the oil comprises about 5% toabout 90% of the reaction mixture by weight. In some embodiments, theshort chain fatty acid esters can be about 10% to about 50% of thereaction mixture by weight. Non-limiting examples of catalysts includebase catalysts, sodium methoxide, acid catalysts including inorganicacids such as sulfuric acid and acidified clays, organic acids such asmethane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid,and acidic resins such as Amberlyst 15. Metals such as sodium andmagnesium, and metal hydrides also are useful catalysts.

Another such chemical modification is hydroxylation, which involves theaddition of water to a double bond resulting in saturation and theincorporation of a hydroxyl moiety. The hydroxylation process provides amechanism for converting one or more fatty acid constituents of aglycerolipid to a hydroxy fatty acid. Hydroxylation can be performed,for example, via the method reported in U.S. Pat. No. 5,576,027.Hydroxylated fatty acids, including castor oil and its derivatives, areuseful as components in several industrial applications, including foodadditives, surfactants, pigment wetting agents, defoaming agents, waterproofing additives, plasticizing agents, cosmetic emulsifying and/ordeodorant agents, as well as in electronics, pharmaceuticals, paints,inks, adhesives, and lubricants. One example of how the hydroxylation ofa glyceride may be performed is as follows: fat may be heated,preferably to about 30-50° C. combined with heptane and maintained attemperature for thirty minutes or more; acetic acid may then be added tothe mixture followed by an aqueous solution of sulfuric acid followed byan aqueous hydrogen peroxide solution which is added in small incrementsto the mixture over one hour; after the aqueous hydrogen peroxide, thetemperature may then be increased to at least about 60° C. and stirredfor at least six hours; after the stirring, the mixture is allowed tosettle and a lower aqueous layer formed by the reaction may be removedwhile the upper heptane layer formed by the reaction may be washed withhot water having a temperature of about 60° C.; the washed heptane layermay then be neutralized with an aqueous potassium hydroxide solution toa pH of about 5 to 7 and then removed by distillation under vacuum; thereaction product may then be dried under vacuum at 100° C. and the driedproduct steam-deodorized under vacuum conditions and filtered at about50° to 60° C. using diatomaceous earth.

Hydroxylation of microbial oils produced by the methods described hereincan be performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,590,113 (Oil-based coatings and ink); U.S. Pat. No. 4,049,724(Hydroxylation process); U.S. Pat. No. 6,113,971 (Olive oil butter);U.S. Pat. No. 4,992,189 (Lubricants and lube additives); U.S. Pat. No.5,576,027 (Hydroxylated milk); and U.S. Pat. No. 6,869,597 (Cosmetics).

Hydroxylated glycerolipids can be converted to estolides. Estolidesconsist of a glycerolipid in which a hydroxylated fatty acid constituenthas been esterified to another fatty acid molecule. Conversion ofhydroxylated glycerolipids to estolides can be carried out by warming amixture of glycerolipids and fatty acids and contacting the mixture witha mineral acid, as described by Isbell et al., JAOCS 71(2):169-174(1994). Estolides are useful in a variety of applications, includingwithout limitation those reported in the following: U.S. Pat. No.7,196,124 (Elastomeric materials and floor coverings); U.S. Pat. No.5,458,795 (Thickened oils for high-temperature applications); U.S. Pat.No. 5,451,332 (Fluids for industrial applications); U.S. Pat. No.5,427,704 (Fuel additives); and U.S. Pat. No. 5,380,894 (Lubricants,greases, plasticizers, and printing inks).

Other chemical reactions that can be performed on microbial oils includereacting triacylglycerols with a cyclopropanating agent to enhancefluidity and/or oxidative stability, as reported in U.S. Pat. No.6,051,539; manufacturing of waxes from triacylglycerols, as reported inU.S. Pat. No. 6,770,104; and epoxidation of triacylglycerols, asreported in “The effect of fatty acid composition on the acrylationkinetics of epoxidized triacylglycerols”, Journal of the American OilChemists' Society, 79:1, 59-63, (2001) and Free Radical Biology andMedicine, 37:1, 104-114 (2004).

The generation of oil-bearing microbial biomass for fuel and chemicalproducts as described above results in the production of delipidatedbiomass meal. Delipidated meal is a byproduct of preparing algal oil andis useful as animal feed for farm animals, e.g., ruminants, poultry,swine and aquaculture. The resulting meal, although of reduced oilcontent, still contains high quality proteins, carbohydrates, fiber,ash, residual oil and other nutrients appropriate for an animal feed.Because the cells are predominantly lysed by the oil separation process,the delipidated meal is easily digestible by such animals. Delipidatedmeal can optionally be combined with other ingredients, such as grain,in an animal feed. Because delipidated meal has a powdery consistency,it can be pressed into pellets using an extruder or expander or anothertype of machine, which are commercially available.

The invention, having been described in detail above, is exemplified inthe following examples, which are offered to illustrate, but not tolimit, the claimed invention.

VII. EXAMPLES Example 1 Methods for Culturing Prototheca

Prototheca strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperatureand 500 ul of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.24 g/L MgSO₄.7H₂O, 0.25 g/L Citric Acid monohydrate,0.025 g/L CaCl₂2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for7 days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 min in a pre-weighed Eppendorf tube. The culture supernatant wasdiscarded and the resulting cell pellet washed with 1 ml of deionizedwater. The culture was again centrifuged, the supernatant discarded, andthe cell pellets placed at −80° C. until frozen. Samples were thenlyophilized for 24 hrs and dry cell weights calculated. Fordetermination of total lipid in cultures, 3 ml of culture was removedand subjected to analysis using an Ankom system (Ankom Inc., Macedon,N.Y.) according to the manufacturer's protocol. Samples were subjectedto solvent extraction with an Amkom XT10 extractor according to themanufacturer's protocol. Total lipid was determined as the difference inmass between acid hydrolyzed dried samples and solvent extracted, driedsamples. Percent oil dry cell weight measurements are shown in Table 8.

TABLE 8 Percent oil by dry cell weight Species Strain % Oil Protothecastagnora UTEX 327 13.14 Prototheca moriformis UTEX 1441 18.02 Protothecamoriformis UTEX 1435 27.17

Microalgae samples from the strains listed in Table 22 above weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centrifuged from liquid cultures 5 minutesat 14,000×g. Cells were then resuspended in sterile distilled water,centrifuged 5 minutes at 14,000×g and the supernatant discarded. Asingle glass bead ˜2 mm in diameter was added to the biomass and tubeswere placed at −80° C. for at least 15 minutes. Samples were removed and150 μl of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pelletswere resuspended by vortexing briefly, followed by the addition of 40 ulof 5M NaCl. Samples were vortexed briefly, followed by the addition of66 μl of 5% CTAB (Cetyl trimethylammonium bromide) and a final briefvortex. Samples were next incubated at 65° C. for 10 minutes after whichthey were centrifuged at 14,000×g for 10 minutes. The supernatant wastransferred to a fresh tube and extracted once with 300 μl ofPhenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugationfor 5 minutes at 14,000×g. The resulting aqueous phase was transferredto a fresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:9) at 10mM stock concentration). This primer sequence runs from position 567-588in Gen Bank accession no. L43357 and is highly conserved in higherplants and algal plastid genomes. This was followed by the addition of0.4 μl primer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:10) at 10mM stock concentration). This primer sequence is complementary toposition 1112-1093 in Gen Bank accession no. L43357 and is highlyconserved in higher plants and algal plastid genomes. Next, 5 μl ofdiluted total DNA and 3.2 μl dH₂O were added. PCR reactions were run asfollows: 98° C., 45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cyclesfollowed by 72° C. for 1 mM and holding at 25° C. For purification ofPCR products, 20 μl of 10 mM Tris, pH 8.0, was added to each reaction,followed by extraction with 40 μl of Phenol:Chloroform:isoamyl alcohol12:12:1, vortexing and centrifuging at 14,000×g for 5 minutes. PCRreactions were applied to S-400 columns (GE Healthcare) and centrifugedfor 2 minutes at 3,000×g. Purified PCR products were subsequently TOPOcloned into PCR8/GW/TOPO and positive clones selected for on LB/Specplates. Purified plasmid DNA was sequenced in both directions using M13forward and reverse primers. In total, twelve Prototheca strains wereselected to have their 23S rRNA DNA sequenced and the sequences arelisted in the Sequence Listing. A summary of the strains and SequenceListing Numbers is included below. The sequences were analyzed foroverall divergence from the UTEX 1435 (SEQ ID NO: 15) sequence. Twopairs emerged (UTEX 329/UTEX 1533 and UTEX 329/UTEX 1440) as the mostdivergent. In both cases, pairwise alignment resulted in 75.0% pairwisesequence identity. The percent sequence identity to UTEX 1435 is alsoincluded below.

Species Strain % nt identity SEQ ID NO. Prototheca kruegani UTEX 32975.2 SEQ ID NO: 11 Prototheca wickerhamii UTEX 1440 99 SEQ ID NO: 12Prototheca stagnora UTEX 1442 75.7 SEQ ID NO: 13 Prototheca moriformisUTEX 288 75.4 SEQ ID NO: 14 Prototheca moriformis UTEX 1439; 100 SEQ IDNO: 15 1441; 1435; 1437 Prototheca wikerhamii UTEX 1533 99.8 SEQ ID NO:16 Prototheca moriformis UTEX 1434 75.9 SEQ ID NO: 17 Prototheca zopfiiUTEX 1438 75.7 SEQ ID NO: 18 Prototheca moriformis UTEX 1436 88.9 SEQ IDNO: 19

Lipid samples from a subset of the above-listed strains were analyzedfor lipid profile using HPLC. Results are shown below in Table 9.

TABLE 9 Diversity of lipid chains in microalgal species Strain C14:0C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 UTEX 0 12.01 0 0 50.3317.14 0 0 0 327 UTEX 1.41 29.44 0.70 3.05 57.72 12.37 0.97 0.33 0 1441UTEX 1.09 25.77 0 2.75 54.01 11.90 2.44 0 0 1435

Algal plastid transit peptides were identified through the analysis ofUTEX 1435 (Prototheca moriformis) or UTEX 250 (Chlorella protothecoides)cDNA libraries as described in Examples 12 and Example 11 below. cDNAsencoding potentially plastid targeted proteins based upon BLAST hithomology to other known plastid targeted proteins were subjected tofurther analysis by the software programs PSORT(http://psort.ims.u-tokyo.ac.jp/form.html), ChloroP(http://www.cbs.dtu.dk/services/ChloroP/) are TargetP(http://www.cbs.dtu.dk/services/TargetP/). Candidate plastid transitpeptides identified through at least one of these three programs werethen PCR amplified from the appropriate genomic DNA. Below is a summaryof the amino acid sequences algal plastid targeting sequences (PTS) thatwere identified from this screen. Also included are the amino acidsequences of plant fatty acyl-ACP thioesterases that are used in theheterologous expression Examples below.

cDNA SEQ ID NO. P. moriformis isopentenyl diphosphate synthase PTS SEQID NO: 127 P. moriformis delta 12 fatty acid desaturase PTS SEQ ID NO:128 P. moriformis stearoyl ACP desaturase PTS SEQ ID NO: 129 C.protothecoides stearoyl ACP desaturase PTS SEQ ID NO: 130 Cupheahookeriana fatty acyl-ACP thioesterase SEQ ID NO: 131 (C8-10)Umbellularia californica fatty acyl-ACP SEQ ID NO: 132 thioesterase(C12) Cinnamomum camphora fatty acyl-ACP SEQ ID NO: 133 thioesterase(C14)

Example 2 Culturing Prototheca on Various Feedstocks

A. Sorghum

The following strains were shown to be capable of utilizing sorghum as asole carbon source: Prototheca moriformis strains UTEX 1435, UTEX 1437,UTEX 288, UTEX 1439, UTEX 1441 and UTEX 1434, and Prototheca stagnorastrain UTEX 1442. The “UTEX” designation indicates the strain numberfrom the algal culture collection of the University of Texas, 1University State A6700, Austin, Tex. 78712-0183.

Pure sorghum was purchased from Maasdam Sorghum Mills (Lynnville, Iowa)with a sugar profile of fructose 21.0% w/w, dextrose 28.0% w/w, sucrose16.0% w/w and maltose <0.5% w/w. The cultures were grown in liquidmedium containing 2%, 5%, or 7% (v/v) pure sorghum (diluted from thepure stock) as the sole carbon source and the cultures were grownheterotrophically in the dark, agitating at ˜350 rpm. Samples from thecultures were pulled at 24, 40, 48, 67 and 89 hours and growth wasmeasured using A750 readings on a spectrophotometer. Growth was observedfor each of the strains tested as shown in FIGS. 1-2.

B. Cellulose

Wet, exploded corn stover, Miscanthus, forage sorghum, beet pulp andsugar cane bagasse were prepared by The National Renewable EnergyLaboratory (Golden, Colo.) by cooking in a 1.4% sulfuric acid solutionand dewatering the resultant slurry. Percent solids were determinedgravimetric ally by drying and were as follows: corn stover, 25% solids;Miscanthus, 28.7% solids; forage sorghum, 26.7% solids; and sugar canebagasse, 26% solids.

100 gram wet samples of exploded cellulosic materials (corn stover orswitch grass) were resuspended in deionized water to a final volume of420 mL and the pH was adjusted to 4.8 using 10N NaOH. For beet pulp, 9.8grams dry solids were brought to 350 mL with deionized water and pH wasadjusted to 4.8 with 10 N NaOH. For all of the above feedstocks,Accellerase 1000 (Genencor, N.Y.) was used at a ratio of 0.25 ml enzymeper gram of dry biomass for saccharification of the cellulosicmaterials. Samples were incubated with agitation (110 rpm) at 50° C. for72 hours. The pH of each of the samples was adjusted to 7.0 with NaOH(with negligible volume change), filter sterilized through a 0.22 μmfilter and used in the processes detailed below. For larger scaleprocesses, the same procedure for saccharification was followed exceptan additional step of tangential flow filtration (TFF) ormicrofiltration step was performed to aid in filter sterilization offeedstocks. A sample from each of the feedstocks prepared was reservedfor determination of glucose and xylose concentration using anHPLC/ELSD-based system or a hexokinase-based kit (Sigma). Additionally,for beet pulp, the material was initially brought to volume as with theother feedstocks, the pH was then adjusted to 4.0 and a pectinasetreatment was carried out at 50° C. for 24 hours. The pH was thenadjusted to 4.8 if no washing steps were conducted or 5.3 if washingsteps were conducted. Enzymatic saccharification was then performed withthe same procedure used for the other feedstocks as described above.

Microalgae Prototheca moriformis strain UTEX 1435 was assessed for itsability to grow on a series of cellulosic feedstocks prepared asdescribed above (corn stover, beet pulp, sorghum cane, Miscanthus andglucose control). The microalgae culture was grown in conditionsdescribed in Example 1 above with the exception of the carbon source.The carbon source was either 4% glucose (for control conditions) or 4%glucose as measured by available glucose in the cellulosic materials.Growth was assessed by A750 readings and the culturing time was 168hours, with A750 readings at 48, 72, 96, 120, 144 and 168 hours afterinitiation of the culture. As can be seen in FIG. 7a , the Protothecamoriformis culture grew best in corn stover. The other cellulosicfeedstocks used, Miscanthus, sorghum cane and beet pulp, all exhibitedinhibition of growth.

Based on the above results with corn stover derived cellulosic sugars,lipid accumulation was also assessed in Prototheca moriformis usingdifferent levels of corn stover derived cellulosic sugars and reagentglucose as a control. Cultures were grown in 18 g/L glucose that wascompletely from corn stover derived cellulosic sugars (100% corn stovercondition in FIG. 7b ), 9 g/L glucose from corn stover derivedcellulosic sugars supplemented with 9 g/L reagent glucose (50% cornstover supplemented with glucose to 18 g/L condition in FIG. 7b ), 9 g/Lglucose from corn stover derived cellulosic sugars (50% corn stover, notsupplemented; glucose at 9 g/L condition in FIG. 7b ) and a controlculture of 42 g/L reagent glucose and 13 g/L reagent xylose forosmolarity control. All cultures were fed with cellulosic sugars tomaintain the glucose concentration at 20 g/L, except for the controlculture, which was fed with reagent glucose to maintain the glucoseconcentration at 20 g/L. Growth was measured based on the dry cellweight of the culture and lipid productivity was determined as a percentdry cell weight. Total lipids were determined gravimetrically using anAnkom acid hydrolysis/solvent extraction system as described in Example1 above.

As can be seen in FIG. 7b , based on biomass accumulation (as measuredby DCW), all concentrations of the corn stover derived cellulosicsout-performed (higher DCW) the control media that was fed glucose alone.Lipid production as a percentage of DCW was also calculated for all ofthe conditions. In addition to the higher biomass accumulation seen forgrowth on corn stover, lipid accumulation was also higher in the cornstover derived cellulosics conditions as compared to the glucose controlcondition. These data demonstrate that, in addition to providingcellulosic derived sugars, corn stover provides additionalnutrients/components that contribute to an increased biomassaccumulation (growth) and increased product yield.

Because the cellulosic feedstocks contain components in addition toglucose, some of these additional components can accumulate toundesirable levels during culture as more cellulosic derived sugars arefed into the culture as the main carbon source (usually, but not limitedto, glucose) is consumed. For example, the xylose present in thecellulosic derived sugar feedstock may build up during the high densitycultivation of microalgae to levels inhibitory to growth and end productproduction. To test the effects of xylose build up during Protothecacultivation, cultures were grown with 4% glucose in the media andsupplemented with 0, 10 g/L, 25 g/L, 50 g/L and 100 g/L xylose. After 6days of culture, growth and lipid accumulation were assessed using themethods described above. As seen in FIG. 7c , surprisingly, the highestconcentrations of xylose tested were not inhibitory to Protothecamoriformis' ability to grow and accumulate lipid, and the cultureactually grew better and accumulated more lipids at the highest xyloseconcentrations. To explore this phenomenon, a similar experiment wascarried out with sucrose, a carbon source which wild type Protothecamoriformis is unable to metabolize. No positive impact was observed withsucrose, suggesting that the increased growth and lipid accumulationseen with xylose is attributable to a mechanism other than the osmoticstress from high concentrations of unmetabolized components in the mediaand is xylose-specific.

In addition to non-metabolized sugars, salts may accumulate toinhibitory levels as a result of concentrating lignocellulosic derivedsugars. Due to the acid hydrolysis step with H₂SO₄ during the typicalpreparation of cellulosic materials followed by neutralization of theacid with NaOH, Na₂SO₄ is formed during the generation oflignocellulosic sugars. To assess the impact of salt concentration ongrowth and lipid production, Prototheca moriformis cultures were grownat Na₂SO₄ concentrations ranging from 0-700 mM in media supplementedwith 4% glucose. As shown in FIG. 7d , a significant inhibition ofgrowth was observed, as measured by DCW accumulation, where Na₂SO₄concentrations exceeded 25 mM, specifically at the 80 mM, 240 mM and 700mM concentrations. In addition, the impact of antifoam P2000 wasassessed in the same test. The antifoam compound had a significant,positive impact on biomass productivity. Lipid productivity was alsoassessed for each condition, and Na2SO4 concentrations above 80 mM,specifically 240 mM and 700 mM, were inhibitory while the addition ofantifoam P2000 significantly increased lipid productivity. Thus, in oneembodiment, the culturing steps of the methods of the present inventioninclude culturing in media containing an antifoaming agent.

Based on the results discussed above and summarized in FIG. 7a ,inhibitors were likely present in the cellulosic feedstocks exhibitingpoor growth. The present invention provides means of removing suchcompounds by washing the materials with hot water (hydrothermaltreatment). FIG. 8 summarizes the growth results, as measured by A750,using sugar derived from cellulosic feedstock with a single hot waterwash. The culture conditions were identical to those used in theprocesses summarized in FIG. 7a . Compared to the results shown in FIG.7a , after just one hot water wash, Prototheca moriformis cultures grewbetter in all cellulosic feedstocks tested, specifically sugar canebagasse, sorghum cane, Miscanthus and beet pulp, as compared to glucosecontrol. Lipid productivity was also assessed in each of the conditions.Except for the beet pulp condition, which was comparable to the glucosecontrol, cultures grown in sugars derived from cellulosic materialssubjected to one hot water wash exhibited better lipid productivity thanthe glucose control.

One potential impact of hydrothermal treatment (hot water washing) ofcellulosic biomass is the removal of furfurals and hydroxymethylfurfurals released by acid explosion of the material. The presence offurfurals and hydroxymethyl furfurals may have contributed to limitedgrowth observed in some of the processes summarized in FIG. 7a . Toassess how hydrothermal treatment affected the levels of furfurals (FA)and hydroxymethyl furfurals (HMF), supernatants resulting from one tothree washes of cellulosic biomass derived from sugarcane bagasse (B),sorghum cane (S), Miscanthus (M) or beet pulp (BP) were assayed for FAand HMF by HPLC. As shown in FIG. 8, FA and HMF levels decreasesignificantly with each washing step. This result is consistent with theobservation that FA and HMF can be inhibitory to microalgal growth (asseen in FIG. 7a ) and that hydrothermal treatment removes thesecompounds and results in improved microalgal growth, even better thanthe growth in the control glucose conditions (as seen in FIG. 8).

The impact on the lipid profile of Prototheca moriformis cultures grownon the various hydrothermally treated lignocellulosic derived sugars wasassessed. Prototheca moriformis cultures were grown on the following4×-washed cellulosic feedstocks: Miscanthus, sugar cane bagasse andsorghum cane, with glucose levels maintained at 20 g/L through feedingof the cellulosic sugars. At the conclusion of the culturing, microalgaebiomass from each condition was analyzed for lipid profile using themethods described in Example 1. The results of the lipid profileanalysis (expressed in Area %) are summarized in Table 10 below. Eachcondition was tested in duplicates, and the results from each of theduplicate test conditions are included. Growth on cellulosic feedstocksresulted in a significant re-distribution in the lipid profile ascompared to the glucose control. For example, there was a significantincrease in C18:0 Area % in all of the cellulosic feedstock conditionsas compared to the glucose control condition.

TABLE 10 Lipid profile of Prototheca moriformis grown on glucose andcellulosics derived sugars. glucose 1 glucose 2 (ctrl) (ctrl) bagasse 1bagasse 2 sorgh 1 sorgh 2 Miscan 1 Miscan 2 C10:0 n.d. n.d. 0.03 0.02n.d. n.d. n.d. n.d. C12:0 0.04 0.05 0.04 0.04 0.05 0.04 0.04 0.04 C14:01.64 1.64 1.07 1.10 1.17 1.14 1.08 1.12 C14:1 0.03 0.04 0.04 0.04 0.060.06 0.03 0.03 C15:0 0.04 0.05 0.07 0.05 0.08 0.08 0.06 0.06 C16:0 26.8026.81 22.32 22.81 22.09 22.19 23.45 23.62 C16:1 0.75 0.82 1.68 1.70 1.922.12 1.38 1.23 C17:0 0.14 0.16 0.28 0.17 0.29 0.27 0.21 0.19 C17:1 0.070.06 0.10 0.10 0.13 0.12 0.10 0.09 C18:0 3.56 3.64 15.88 10.40 15.3012.37 10.15 8.69 C18:1 54.22 54.01 49.87 53.87 49.35 50.80 54.05 55.26C18:2 11.23 11.11 6.54 7.91 7.47 8.80 7.71 7.88 C18:3 0.84 0.85 0.390.56 0.47 0.53 0.56 0.60 alpha C20:0 0.31 0.30 0.85 0.63 0.76 0.69 0.630.56 C20:1 0.15 0.15 0.33 0.28 0.32 0.32 0.27 0.25 C20:3 0.06 0.06 0.130.12 0.14 0.12 0.11 0.11 C24:0 0.12 0.12 0.22 0.19 0.22 0.20 0.18 0.15n.d. denotes none detected

Cellulosic sugar stream was generated from exploded corn stover,saccharified using Accellerase enzyme and concentrated using vacuumevaporation. This sugar stream was tested in Prototheca moriformisgrowth assays at a 4% glucose concentration. The results of the growthassays showed very poor growth and the cellulosic sugar stream wastested for conductivity (salt content). The conductivity was very high,far greater than 700 mM sodium equivalents, a level that was shown to beinhibitory to growth as described above and shown in FIG. 7d . Methodsof the invention include methods in which salt is reduced or removedfrom lignocellulosic derived sugars prior to utilizing these feedstocksin the production of lignocellulosic derived microalgal oil.Surprisingly, however, one cannot use resins to desalt concentratedsugar streams, one must first dilute the concentrated sugar stream. Todemonstrate this embodiment of the invention, cellulosic sugars derivedfrom corn stover material were diluted eight-fold prior to removingcontaminating salts with the resin. The initial conductivity of theconcentrated starting material was 87 mS/cm while that of the eight-folddiluted stream was 10990 μS/cm at a pH of 5.61. Previous studies hadindicated that failure to dilute the concentrated sugar stream prior tode-ionization resulted in an inability to remove salts quantitatively aswell as a significant loss of glucose from the sugar stream. Threedifferent bed volumes of IEX resin (DOWEX Marathon MR3) were used (1:2,1:4 and 1:10). Table 11 summarize results demonstrating the ability of amixed bed ion exchange (IEX) resin to reduce salts (as measured byconductivity) significantly in a previously concentrated corn stoverderived cellulosic sugar stream in diluted feedstocks.

TABLE 11 Ability of IEX resin to reduce salts. Calculated conductivitypost Conductivity post- deionization and 8x Na⁺ equivalents pH post-deionization re-concentration (based on std curve) Bed volumeresin:cellulosics deionization (μS/cm) (μS/cm) in mM 1:2 3.1 74 592 7.421:4 3.1 97 776 9.7  1:10 5.25 6320 50560 634

A process employing a 1:4 bed volume:cellulosic feedstock andre-concentration of the material eight-fold would result in a sodiumconcentration is well within the range for normal biomass and lipidaccumulation. Alternatively, deionization or salt removal can beperformed prior to saccharification or after saccharification, butbefore concentration of the sugar stream. If salt removal is performedbefore the concentration of the sugar stream, a dilution step of thesugar stream before salt removal would likely not be necessary.

This example demonstrates the efficacy of washing of exploded cellulosicmaterial for the use in cellulosic oil production. As described above,concentration of cellulosically derived sugars without the removal ofsalts (inherent to the production of exploded cellulosic material andsubsequent treatment) results in less than optimal fermentations. Thematerials treated in the process described below were of the appropriatepH for subsequent saccharifaication. In addition, the conductivity ofthis material was significantly reduced (over 100 fold) from thestarting feedstock. Therefore, the subsequent concentrated sugars to beused in fermentations were not inhibitory due to the presence ofexcessive salts. An additional advantage is seen by the removal offurfurals from the cellulosic material. Any xylose or glucose removed inthe hemicellulosic fraction can either be discarded or preferablyre-concentrated to be used in fermentations.

Wet, exploded sugar cane bagasse (NREL, Colorado) with an initialstarting mass of 65 kg wet weight and conductivity of 15,000 μS/cm, pH2.4 was brought to 128 kg with deionized water and the pH adjusted to4.6 with 10 N NaOH, making the resulting conductivity 6,800 μS/cm). Thepercent solids were assessed by removal of an aliquot of the suspendedmaterials to a tared (weight=t) aluminum pan, recording the wet weight(weight=w) followed by drying for three hours at 110° C. After dryingsamples were removed to a desiccator and allowed to come to roomtemperature (25° C.) at which point, they were weighed again (weight=d).Percent solids were calculated as: % solids=[(d−t/w−t)]×100.Conductivities were measured on a Thermo Electron Orion 3 StarConductivity meter.

The sugar cane bagasse was washed in a semi-continuous fashion bycontinuously mixing the cellulosic slurry (initial percent solids of8.2%) at a temperature of 50° C. in a stainless steel reactor (150 Lcapacity). Cellulosics were discharged from the reactor vessel via arotary load pump at a flow rate of 1.9-3.8 kg/min to a Sharples Model660 decanter centrifuge. Liquid permeate was retained batch wise (ca.35-175 kg aliquots, see Table 12 below) and homogenous aliquots removedfor assessment of total sugars (glucose and xylose) and percent solidsas described in Table 12. Conductivity and pH of the cellulosic materialwere controlled via the addition of de-ionized water and 10 N NaOH,respectively. Samples 1-10 in Table 12 represent decanted centrifugepermeate, and as such, solids and sugars present in these fractions areremoved from the final, washed cellulosic materials. A mass balancecalculation of total solids compared to solids removed minus solids lostplus final solids for saccharification, resulted in a 99% recovery inthe above process. FIG. 8 summarizes the furfural and hydroxymethylfurfurals concentration (mg/L) in each of the 11 centrifuge permeatescollected and described in Table 12. These data demonstrate a clearremoval of furfurals and hydroxymethyl furfurals from the sugar canebagasse.

TABLE 12 Mass balance for semi-continuous hydrothermal treatment ofsugar cane bagasse. total Con- total glu- duc- xylose cose tivityremoved removed Sample kg (wet) kg (dry) pH μS/cm (g) (g) 1 (initial 12810.50 4.60 6,880 0 0 material) 2 81.8 2.03 3,280 1030.68 286.3 3 76.50.49 2,500 298.35 76.50 4 106 0.41 254.40 63.60 5 173.9 0.30 3.74 1,260226.07 69.56 6 101.8 0.08 4.40 791 71.26 20.36 7 110.6 0.04 4.86 32744.24 0 8 77.2 0 0 0 9 108.6 0.02 4.7 221 0 0 10 101.5 0 0 0 11 34.8 04.7 146 0 0 Solids removed 3.37 (samples 1-10) lost in process Totalxylose 1925.00 removed Total glucose 516.32 removed Final solids for7.03 saccharification

In another demonstration of the ability of Prototheca to utilizecellulosic-derived feedstock, Prototheca moriformis (UTEX 1435) wascultivated in three-liter bioreactors using cellulosic derived sugar asa fixed carbon feedstock. The inoculum was prepared from cryopreservedcells, which were thawed at room temperature and 1 mL of cells wereadded to 300 mL of inoculum medium based on the basal microalgae mediumdescribed in Example 1 with 1 g/L (NH₄)₂SO₄, 4 g/L yeast extract and atrace element solution, plus 4% glucose and grown for 1 day at 28° C.with agitation (200 rpm). This culture was used to inoculate athree-liter bioreactor containing 1 L medium plus 0.26 mL of Antifoam204 (Sigma, USA). The fermentor was controlled at 28° C. and pH wasmaintained at 6.8 by addition of KOH. Dissolved oxygen was maintained at30% saturation by cascading agitation and airflow. Cellulosic sugarfeedstock from corn stover was fed to the culture to maintain 0-10 g/Lglucose. Desalination of cellulosic sugar feedstocks to less than 300 mMsalt was essential to assure similar dry cell weight and lipidaccumulation performance as compared to purified sugar feedstockcontrols. Desalination of the cellulosic sugar feedstock was performedusing the methods described above. Fermentor samples were removed tomonitor fermentation performance. Cell mass accumulation was monitoredby optical density and dry cell weight. Glucose, xylose, ammonia,potassium, sodium and furfural concentrations were also determined andmonitored throughout the fermentation time course. Lipid concentrationwas determined by gravimetric methods discussed above.

Example 3 Methods for Transforming Prototheca

A. General Method for Biolistic Transformation of Prototheca

S550d gold carriers from Seashell Technology were prepared according tothe protocol from manufacturer. Linearized plasmid (20 μg) was mixedwith 50 μl of binding buffer and 60 μl (30 mg) of S550d gold carriersand incubated in ice for 1 min Precipitation buffer (100 μl) was added,and the mixture was incubated in ice for another 1 min. After vortexing,DNA-coated particles were pelleted by spinning at 10,000 rpm in anEppendorf 5415C microfuge for 10 seconds. The gold pellet was washedonce with 500 μl of cold 100% ethanol, pelleted by brief spinning in themicrofuge, and resuspended with 50 μl of ice-cold ethanol. After a brief(1-2 sec) sonication, 10 μl of DNA-coated particles were immediatelytransferred to the carrier membrane.

Prototheca strains were grown in proteose medium (2 g/L yeast extract,2.94 mM NaNO3, 0.17 mM CaCl2.2H2O, 0.3 mM Mg5O4.7H2O, 0.4 mM K2HPO4,1.28 mM KH2PO4, 0.43 mM NaCl) on a gyratory shaker until it reaches acell density of 2×10⁶ cells/ml. The cells were harvested, washed oncewith sterile distilled water, and resuspended in 50 μl of medium. 1×10⁷cells were spread in the center third of a non-selective proteose mediaplate. The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used,and the plates are placed 9 and 12 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 h. Uponrecovery, the cells were scraped from the plates with a rubber spatula,mixed with 100 μl of medium and spread on plates containing theappropriate antibiotic selection. After 7-10 days of incubation at 25°C., colonies representing transformed cells were visible on the platesfrom 1100 and 1350 psi rupture discs and from 9 and 12 cm distances.Colonies were picked and spotted on selective agar plates for a secondround of selection.

B. Transformation of Prototheca with G418 Resistance Gene

Prototheca moriformis and other Prototheca strains sensitive to G418 canbe transformed using the methods described below. G418 is anaminoglycoside antibiotic that inhibits the function of 80S ribosomesand thereby inhibits protein synthesis. The corresponding resistancegene functions through phosphorylation, resulting in inactivation ofG418. Prototheca strains UTEX 1435, UTEX 1439 and UTEX 1437 wereselected for transformation. All three Prototheca strains were genotypedusing the methods described above. All three Prototheca strains hadidentical 23s rRNA genomic sequences (SEQ ID NO:15).

All transformation cassettes were cloned as EcoRI-SacI fragments intopUC19. Standard molecular biology techniques were used in theconstruction of all vectors according to Sambrook and Russell, 2001. TheC. reinhardtii beta-tubulin promoter/5′UTR was obtained from plasmidpHyg3 (Berthold et al., (2002) Protist: 153(4), pp 401-412) by PCR as anEcoRI-AscI fragment. The Chlorella vulgaris nitrate reductase 3′UTR wasobtained from genomic DNA isolated from UTEX strain 1803 via PCR usingthe following primer pairs:

Forward: (SEQ ID NO: 35) 5′TGACCTAGGTGATTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTA TCG 3′ Reverse:(SEQ ID NO: 36) 5′ CTACGAGCTCAAGCTTTCCATTTGTGTTC CCATCCCACTACTTCC 3′

The Chlorella sorokiniana glutamate dehydrogenase promoter/UTR wasobtained via PCR of genomic DNA isolated from UTEX strain 1230 via PCRusing the following primer pairs:

Forward: (SEQ ID NO: 37) 5′ GATCAGAATTCCGCCTGCAACGCAAGG GCAGC 3′Reverse: (SEQ ID NO: 38) 5′ GCATACTAGTGGCGGGACGGAGAGA GGGCG 3′

Codon optimization was based on the codons in Table 1 for Protothecamoriformis. The sequence of the non-codon optimized neomycinphosphotransferase (nptII) cassette was synthesized as an AscI-XhoIfragment and was based on upon the sequence of Genbank Accession No.YP_788126. The codon optimized nptII cassette was also based on thisGenbank Accession number.

The three Prototheca strains were transformed using biolistic methodsdescribed above. Briefly, the Prototheca strains were grownheterophically in liquid medium containing 2% glucose until they reachedthe desired cell density (1×10⁷ cells/mL to 5×10⁷ cells/mL). The cellswere harvested, washed once with sterile distilled water and resuspendedat 1×10⁸ cells/mL. 0.5 mL of cells were then spread out on anon-selective solid media plate and allowed to dry in a sterile hood.The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery System (BioRad). The cells were allowed to recover at 25° C.for 24 hours. Upon recovery, the cells were removed by washing plateswith 1 mL of sterile media and transferring to fresh plates containing100 μg/mL G418. Cells were allowed to dry in a sterile hood and colonieswere allowed to form on the plate at room temperature for up to threeweeks. Colonies of UTEX 1435, UTEX 1439 and UTEX 1437 were picked andspotted on selective agar plates for a second round of selection.

A subset of colonies that survived a second round of selection describedabove, were cultured in small volume and genomic DNA and RNA wereextracted using standard molecular biology methods. Southern blots weredone on genomic DNA extracted from untransformed (WT), the transformantsand plasmid DNA. DNA from each sample was run on 0.8% agarose gels afterthe following treatments: undigested (U), digested with AvrII (A),digested with NcoI (N), digested with SacI (S). DNA from these gels wasblotted on Nylon+ membranes (Amersham). These membranes were probed witha fragment corresponding to the entire coding region of the nptII gene(NeoR probe). FIG. 4 shows maps of the cassettes used in thetransformations. FIG. 5 shows the results of Southern blot analysis onthree transformants (all generated in UTEX strain 1435) (1, 2, and 3)transformed with either the beta-tubulin::neo::nit (SEQ ID NO: 39)(transformants 1 and 2) or glutamate dehydrogenase:neo:nit (SEQ ID NO:40) (transformant 3). The glutamate dehydrogenase:neo:nit transformingplasmid was run as a control and cut with both NcoI and SacI. AvrII doesnot cut in this plasmid. Genomic DNA isolated from untransformed UTEXstrain 1435 shows no hybridization to the NeoR probe.

Additional transformants containing the codon-optimized glutamatedehydrogenase:neo:nit (SEQ ID NO: 41) and codon-optimizedβ-tubulin::neo::nit (SEQ ID NO:42) constructs were picked and analyzedby Southern blot analysis. As expected, only digests with SacI showlinearization of the transforming DNA. These transformation events areconsistent with integration events that occur in the form of oligomersof the transforming plasmid. Only upon digestion with restrictionenzymes that cut within the transforming plasmid DNA do these moleculescollapse down the size of the transforming plasmid.

Southern blot analysis was also performed on transformants generatedupon transformation of Prototheca strains UTEX 1437 and UTEX 1439 withthe glutamate dehydrogenase::neo::nit cassette. The blot was probed withthe NeoR probe probe and the results are similar to the UTEX 1435transformants. The results are indicative of integration eventscharacterized by oligomerization and integration of the transformingplasmid. This type of integration event is known to occur quite commonlyin Dictyostelium discoideum (see, for example, Kuspa, A. and Loomis, W.(1992) PNAS, 89:8803-8807 and Morio et al., (1995) J. Plant Res.108:111-114).

To further confirm expression of the transforming plasmid, Northern blotanalysis and RT-PCR analysis were performed on selected transformants.RNA extraction was performed using Trizol Reagent according tomanufacturer's instructions. Northern blot analysis were run accordingto methods published in Sambrook and Russel, 2001. Total RNA (15 μg)isolated from five UTEX 1435 transformants and untransformed UTEX 1435(control lanes) was separated on 1% agarose-formaldehyde gel and blottedon nylon membrane. The blot was hybridized to the neo-non-optimizedprobe specific for transgene sequences in transformants 1 and 3. The twoother transformants RNAs express the codon-optimized version of theneo-transgene and, as expected, based on the sequence homology betweenthe optimized and non-optimized neo genes, showed significantly lowerhybridization signal.

RNA (1 μg) was extracted from untransformed Prototheca strain UTEX 1435and two representative UTEX 1435 transformants and reverse transcribedusing an oligio dT primer or a gene specific primer. Subsequently thesecDNAs (in duplicate) were subjected to qPCR analysis on ABI VeritiThermocycler using SYBR-Green qPCR chemistry using the following primers(nptII):

(SEQ ID NO: 43) Forward: 5′ GCCGCGACTGGCTGCTGCTGG 3′ (SEQ ID NO: 44)Reverse: 5′ AGGTCCTCGCCGTCGGGCATG 3′

Possible genomic DNA contamination was ruled out by a no reversetranscriptase negative control sample. The results indicated that theNeoR genes used to transform these strains is actively transcribed inthe transformants.

C. Transformation of Prototheca with Secreted Heterologous SucroseInvertase

All of the following experiments were performed using liquid medium/agarplates based on the basal medium described in Ueno et al., (2002) JBioscience and Bioengineering 94(2):160-65, with the addition of traceminerals described in U.S. Pat. No. 5,900,370, and 1×DAS VitaminCocktail (1000× solution): tricine: 9 g, thiamine HCL: 0.67 g, biotin:0.01 g, cyannocobalamin (vitamin B12): 0.008 g, calcium pantothenate:0.02 g and p-aminobenzoic acid: 0.04 g).

Two plasmid constructs were assembled using standard recombinant DNAtechniques. The yeast sucrose invertase genes (one codon optimized andone non-codon optimized), suc2, were under the control of the Chlorellareinhardtii beta-tubulin promoter/5′UTR and had the Chlorella vulgarisnitrate reductase 3′UTR. The sequences (including the 5′UTR and 3′UTRsequences) for the non-codon optimized (Crβ-tub::NCO-suc2::CvNitRed)construct, SEQ ID NO: 57, and codon optimized(Crβ-tub::CO-suc2::CvNitRed) construct, SEQ ID NO: 58, are listed in theSequence Listing. Codon optimization was based on Table 1 for Protothecasp. FIG. 6 shows a schematic of the two constructs with the relevantrestriction cloning sites and arrows indicating the direction oftranscription. Selection was provided by Neo R (codon optimized usingTable 1).

Preparation of the DNA/gold microcarrier: DNA/gold microcarriers wereprepared immediately before use and stored on ice until applied tomacrocarriers. The plasmid DNA (in TE buffer) was added to 50 μl ofbinding buffer. Saturation of the gold beads was achieved at 15 μgplasmid DNA for 3 mg gold carrier. The binding buffer and DNA were mixedwell via vortexing. The DNA and binding buffer should be pre-mix priorto gold addition to ensure uniformed plasmid binding to gold carrierparticles. 60 μl of S550d (Seashell Technologies, San Diego, Calif.)gold carrier was added to the DNA/binding buffer mixture. For a goldstock at 50 mg/ml, addition of 60 μl results in an optimal ratio of 15μg DNA/3 mg gold carrier. The gold carrier/DNA mixture was allowed toincubate on ice for 1 minute and then 100 μl of precipitation buffer wasadded. The mixture was allowed to incubate again on ice for 1 minute andthen briefly vortexed and centrifuged at 10,000 rpm at room temperaturefor 10 seconds to pellet the gold carrier. The supernatant was carefullyremoved with a pipette and the pellet was washed with 500 μl of ice cold100% ethanol. The gold particles were re-pelleted by centrifuging againat 10,000 rpm for 10 seconds. The ethanol was removed and 50 μl of icecold ethanol was added to the gold mixture. Immediately prior toapplying the gold to macrocarriers, the gold/ethanol was resuspendedwith a brief 1-2 second pulse at level 2 on a MISONIX sonicator usingthe micro tip. Immediately after resuspension, 10 μl of the dispersedgold particles was transferred to the macrocarrier and allowed to dry ina sterile hood.

The two Prototheca moriformis strains (UTEX 1435 and 1441) were grownheterotrophically in liquid medium containing 2% glucose fromcryopreserved vials. Each strain was grown to a density of 10⁷ cells/ml.This seed culture was then diluted with fresh media to a density of 10⁵cells/ml and allowed to grow for 12-15 hours to achieve a final celldensity of approximately 10⁶ cells/ml. The microalgae were aliquotedinto 50 ml conical tubes and centrifuged for 10 minutes at 3500 rpm. Thecells were washed with fresh medium and centrifuged again for 10 minutesat 3500 rpm. The cells were then resuspended at a density of 1.25×10⁸cells/ml in fresh medium.

In a sterile hood, 0.4 ml of the above-prepared cells were removed andplaced directly in the center of an agar plate (without selectionagent). The plate was gently swirled with a level circular motion toevenly distribute the cells to a diameter of no more than 3 cm. Thecells were allowed to dry onto the plates in the sterile hood forapproximately 30-40 minutes and then were bombarded at a rupture diskpressure of 1350 psi and a plate to macrocarrier distance of 6 cm. Theplates were then covered and wrapped with parafilm and allowed toincubate under low light for 24 hours.

After the 24 hour recovery, 1 ml of sterile medium (with no glucose) wasadded to the lawn of cells. The cells were resuspended using a sterileloop, applied in a circular motion to the lawn of cells and theresuspended cells were collected using a sterile pipette. The cells werethen plated onto a fresh agar plate with 2% glucose and 100 μg/ml G418.The appearance of colonies occurred 7-12 days after plating. Individualcolonies were picked and grown in selective medium with 2% glucose and100 μg/ml G418. The wildtype (untransformed) and transgenic cells werethen analyzed for successful introduction, integration and expression ofthe transgene.

Genomic DNA from transformed Prototheca moriformis UTEX 1435 and 1441and their wildtype (untransformed) counterparts were isolated usingstandard methods. Briefly, the cells were centrifuged for 5 minutes at14,000 rpm in a standard table top Eppendorf centrifuge (model 5418) andflash frozen prior to DNA extraction. Cell pellets were lysed by adding200 uL of Lysis buffer (100 mM Tris HCl, pH 8.0, 1% Lauryl Sarcosine, 50mM NaCl, 20 mM EDTA, 0.25 M sucrose, 0.5 mg/ml RNase A) for every100-200 mg of cells (wet weight) and vortexing for 30-60 seconds. Cetyltrimethyammonium bromide (CTAB) and NaCl were brought to 1% and 1 M,respectively, and cell extracts were incubated at 60-65° C. for 10minutes. Subsequently, extracts were clarified via centrifugation at14,000 rpm for 10 minutes and the resulting supernatant was extractedwith an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).Samples were then centrifuged for 5 minutes at 14,000 rpm and theaqueous phase removed. DNA was precipitated with 0.7 volumes ofisopropanol. DNA was pelleted via centrifugation at 14,000 rpm for 10minutes and washed twice with 80% ethanol, and once with ethanol. Afterdrying, DNA was resuspended in 10 mM Tris HCl, pH 8.0 and DNAconcentrations were determined by using PicoGreen fluorescencequantification assay (Molecular Probes).

RNA from transformed Prototheca moriformis UTEX 1435 and 1441 and theirwildtype (untransformed) counterparts were isolated using standardmethods. Briefly, the cells were centrifuged for 5 minutes at 14,000 rpmin a standard table top Eppendorf centrifuge (model 5418) and flashfrozen before RNA extraction. Cell pellets were lysed by addition of 1mL of Trizol reagent (Sigma) for every 100 mg of cells (wet weight) andby vortexing for 1-2 minutes. Samples were incubated at room temperaturefor 5 minutes and subsequently adjusted with 200 uL of chloroform per 1mL of Trizol reagent. After extensive shaking, cells were incubated atroom temperature for 15 minutes and then subjected to centrifugation at14000 rpm for 15 minutes in a refrigerated table top microcentrifuge.RNA partitioning to the upper aqueous phase was removed and precipitatedby addition of isopropanol (500 uL per 1 ml of Trizol reagent). RNA wascollected by centrifugation for 10 minutes and the resulting pelletwashed twice with 1 mL of 80% ethanol, dried, and resuspended in RNAsefree water. RNA concentration was estimated by RiboGreen fluorescencequantification assay (Molecular Probes).

Expression of neomycin phophotransferase gene conferring G418 antibioticresistance and yeast invertase was assayed in non-transformed Protothecamoriformis UTEX 1435 and 1441 and transformants T98 (UTEX 1435transformant) and T97 (UTEX 1441 transformant) using reversetranscription quantitative PCR analysis (RT-qPCR). 20 ng total RNA(isolated as described above) was subjected to one step RT-qPCR analysisusing iScript SYBR Green RT-PCR kit (BioRad Laboratories) and primerpairs targeting the neomycin resistance gene (forward primer 5′CCGCCGTGCTGGACGTGGTG 3′ and reverse primer 5′ GGTGGCGGGGTCCAGGGTGT 3′;SEQ ID NOs: 65 and 66, respectively) and suc2 invertase transcripts(forward primer 5′ CGGCCGGCGGCTCCTTCAAC 3′ and reverse primer 5′GGCGCTCCCGTAGGTCGGGT 3′; SEQ ID NO: 67 and 68, respectively). Endogenousbeta-tubulin transcripts served as an internal positive control for PCRamplification and as a normalization reference to estimate relativetranscript levels.

Both codon optimized and non-codon optimized constructs were transformedinto UTEX 1435 and 1441 Prototheca moriformis cells as described above.Initially, transformants were obtained with both constructs and thepresence of the transgene was verified by Southern blot analysisfollowed by RTPCR to confirm the presence of the DNA and mRNA from thetransgene. For the Southern blot analysis, genomic DNA isolated asdescribed above was electrophoresed on 0.7% agarose gels in 1×TAEbuffer. Gells were processed as described in Sambrook et al. (MolecularCloning; A Laboratory Manual, 2^(nd) Edition. Cold Spring HarborLaboratory Press, 1989). Probes were prepared by random priming andhybridizations carried out as described in Sambrook et al. Transformantsfrom both the codon optimized and the non-codon optimized constructsshowed the presence of the invertase cassette, while the non-transformedcontrol was negative. Invertase mRNA was also detected in transformantswith both the codon optimized and non-codon optimized constructs.

To confirm that the transformants were expressing an active invertaseprotein, the transformants were plated on sucrose plates. Thetransformants containing the non-codon optimized cassette failed to growon the sucrose containing plates, indicating that, while the gene andthe mRNA encoding the SUC2 protein were present, the protein was either(1) not being translated, or (2) being translated, but not accumulatingto levels sufficient to allow for growth on sucrose as the sole carbonsource. The transformants with the codon optimized cassette grew on thesucrose containing plates. To assess the levels of invertase beingexpressed by these transformants, two clones (T98 and T97) weresubjected to an invertase assay of whole cells scraped from solid mediumand direct sampling and quantitation of sugars in the culturesupernatants after 48 hours of growth in liquid medium containing 2%sucrose as the sole carbon source.

For the invertase assay, the cells (T98 and T97) were grown on platescontaining 2% sucrose, scraped off and assayed for invertase activity.10 μl of the scraped cells was mixed with 40 μl of 50 mM NaOAc pH 5.1.12.5 μl of 0.5M sucrose was added to the cell mixture and incubated at37° C. for 10-30 minutes. To stop the reaction, 75 μl of 0.2M K₂HPO₄ wasadded. To assay for glucose liberated, 500 μl of reconstituted reagent(glucose oxidase/peroxidase+o-Dianisidine) from Sigma (GAGO-20 assaykit) was added to each tube and incubated at 37° C. for 30 minutes. Aglucose standard curve was also created at this time (range: 25 μg to0.3 μg glucose). After incubation, 500 μl of 6N HCl was added to stopthe reaction and to develop the color. The samples were read at 540 nm.The amount of glucose liberated was calculated from the glucose standardcurve using the formula y=mx+c, where y is the 540 nm reading, and x isμg of glucose. Weight of glucose was converted to moles of glucose, andgiven the equimolar relationship between moles of sucrose hydrolyzed tomoles of glucose generated, the data was expressed as nmoles of sucrosehydrolyzed per unit time. The assay showed that both T98 and T97 cloneswere able to hydrolyze sucrose, indicating that a functional sucroseinvertase was being produced and secreted by the cells.

For the sugar analysis on liquid culture media after 48 hours of algalgrowth, T97 and T98 cells were grown in 2% sucrose containing medium for48 hours and the culture media were processed for sugar analysis.Culture broths from each transformant (and negative non-transformed cellcontrol) were centrifuged at 14,000 rpm for 5 minutes. The resultingsupernatant was removed and subjected to HPLC/ELSD (evaporative lightscattering detection). The amount of sugar in each sample was determinedusing external standards and liner regression analysis. The sucroselevels in the culture media of the transformants were very low (lessthan 1.2 g/L, and in most cases 0 g/L). In the negative controls, thesucrose levels remained high, at approximately 19 g/L after 48 hours ofgrowth.

These results were consistant with the invertase activity results, andtaken together, indicated that the codon optimized transformants, T97and T98, secreted an active sucrose invertase that allowed themicroalgae to utilize sucrose as the sole carbon source in contrast to(1) the non-codon optimized transformants and (2) the non-transformedwildtype microalgae, both of which could not utilize sucrose as the solecarbon source in the culture medium.

Prototheca moriformis strains, T98 and T97, expressing a functional,secreted sucrose invertase (SUC2) transgene were assayed for growth andlipid production using sucrose as the sole carbon source.

Wild type (untransformed), T98 and T97 strains were grown in growthmedia (as described above) containing either 4% glucose or 4% sucrose asthe sole carbon source under heterotrophic conditions for approximately6 days. Growth, as determined by A750 optical density readings weretaken of all four samples every 24 hours and the dry cell weight of thecultures and lipid profiles were determined after the 6 days of growth.The optical density readings of the transgenic strains grown in both theglucose and sucrose conditions were comparable to the wildtype strainsgrown in the glucose conditions. These results indicate that thetransgenic strains were able to grow on either glucose or sucrose as thesole carbon source at a rate equal to wildtype strains in glucoseconditions. The non-transformed, wildtype strains did not grow in thesucrose-only condition.

The biomass for the wildtype strain grown on glucose and T98 straingrown on sucrose was analyzed for lipid profile. Lipid samples wereprepared from dried biomass (lyophilized) using an Acid HydrolysisSystem (Ankom Technology, NY) according to manufacturer's instructions.Lipid profile determinations were carried as described in Example 4. Thelipid profile for the non-transformed Prototheca moriformis UTEX 1435strain, grown on glucose as the sole carbon source and two colonal T98strains (UTEX 1435 transformed with a sucrose invertase transgene),grown on sucrose as the sole carbon source, are disclosed in Table 13(wildtype UTEX 1435 and T98 clone 8 and clone 11 below. C:19:0 lipid wasused as an internal calibration control.

TABLE 13 Lipid profile of wildtype UTEX 1435 and UTEX 1435 clones withsuc2 transgene. wildtype (Area T98 clone 11 T98 clone 8 Name %-ISTD)(Area %-ISTD) (Area %-ISTD) C 12:0 0.05 0.05 0.05 C 14:0 1.66 1.51 1.48C 14:1 0.04 nd nd C 15:0 0.05 0.05 0.04 C 16:0 27.27 26.39 26.50 C 16:10.86 0.80 0.84 C 17:0 0.15 0.18 0.14 C 17:1 0.05 0.07 0.05 C 18:0 3.354.37 4.50 C 18:1 53.05 54.48 54.50 C 18:2 11.79 10.33 10.24 C 19:0(ISTD) — — — C 18:3 alpha 0.90 0.84 0.81 C 20:0 0.32 0.40 0.38 C 20:10.10 0.13 0.12 C 20:1 0.04 0.05 0.04 C 22:0 0.12 0.16 0.12 C 20:3 0.070.08 0.07 C 24:0 0.12 0.11 0.10 nd — denotes none detected

Oil extracted from wildtype Prototheca moriformis UTEX 1435 (via solventextraction or using an expeller press (see methods in Example 44 above)was analyzed for carotenoids, chlorophyll, tocopherols, other sterolsand tocotrienols. The results are summarized below in Table 14.

TABLE 14 Carotenoid, chlorophyll, tocopherol/sterols and tocotrienolanalysis in oil extracted from Prototheca moriformis (UTEX 1435).Pressed oil Solvent extracted (mcg/ml) oil (mcg/ml) cis-Lutein 0.0410.042 trans-Lutein 0.140 0.112 trans-Zeaxanthin 0.045 0.039cis-Zeaxanthin 0.007 0.013 t-alpha-Crytoxanthin 0.007 0.010t-beta-Crytoxanthin 0.009 0.010 t-alpha-Carotene 0.003 0.001c-alpha-Carotene none detected none detected t-beta-Carotene 0.010 0.0099-cis-beta-Carotene 0.004 0.002 Lycopene none detected none detectedTotal Carotenoids 0.267 0.238 Chlorophyll <0.01 mg/kg <0.01 mg/kgTocopherols and Sterols Pressed oil Solvent extracted (mg/100 g) oil(mg/100 g) gamma Tocopherol 0.49 0.49 Campesterol 6.09 6.05 Stigmasterol47.6 47.8 Beta-sitosterol 11.6 11.5 Other sterols 445 446 TocotrienolsPressed oil Solvent extracted (mg/g) oil (mg/g) alpha Tocotrienol 0.260.26 beta Tocotrienol <0.01 <0.01 gamma Tocotrienol 0.10 0.10 detalTocotrienol <0.01 <0.01 Total Tocotrienols 0.36 0.36

The ability of using sucrose as the sole carbon source as the selectionfactor for clones containing the suc2 transgene construct instead ofG418 (or another antibiotic) was assessed using the positive suc2 genetransformants. A subset of the positive transformants was grown onplates containing sucrose as the sole carbon source and withoutantibiotic selection for 24 doublings. The clones were then challengedwith plates containing glucose as the sole carbon source and G418. Therewas a subset of clones that did not grow on the glucose+G418 condition,indicating a loss of expression of the transgene. An additionalexperiment was performed using a plate containing sucrose as the solecarbon source and no G418 and streaking out a suc2 transgene expressingclone on one half of the plate and wild-type Prototheca moriformis onthe other half of the plate. Growth was seen with both the wild-type andtransgene-containing Prototheca moriformis cells. Wild-type Protothecamoriformis has not demonstrated the ability to grow on sucrose,therefore, this result shows that unlike antibiotic resistance, the useof sucrose/invertase selection is not cell-autonomous. It is very likelythat the transformants were secreting enough sucrose invertase into theplate/media to support wildtype growth as the sucrose was hydrolyzedinto fructose and glucose.

Example 4 Recombinant Prototheca with Exogenous TE Gene

As described above, Prototheca strains can be transformed with exogenousgenes. Prototheca moriformis (UTEX 1435) was transformed, using methodsdescribed above, with either Umbellularia californica C12 thioesterasegene or Cinnamomum camphora C14 thiotesterase gene (both codon optimizedaccording to Table 1). Each of the transformation constructs contained aChlorella sorokiniana glutamate dehydrogenase promoter/5′UTR region (SEQID NO: 69) to drive expression of the thioesterase transgene. Thethioesterase transgenes coding regions of Umbellularia californica C12thioesterase (SEQ ID NO: 70) or Cinnamomum camphora C14 thioesterase(SEQ ID NO: 71), each with the native putative plastid targetingsequence Immediately following the thioesterase coding sequence is thecoding sequence for a c-terminal 3×-FLAG tag (SEQ ID NO: 72), followedby the Chlorella vulgaris nitrate reductase 3′UTR (SEQ ID NO: 73). Adiagram of the thioesterase constructs that were used in the Protothecamoriformis transformations is shown in FIG. 9.

Preparation of the DNA, gold microcarrier and Prototheca moriformis(UTEX 1435) cells were performed using the methods described above inExample 3. The microalgae were bombarded using the gold microcarrier—DNAmixture and plated on selection plates containing 2% glucose and 100μg/ml G418. The colonies were allowed to develop for 7 to 12 days andcolonies were picked from each transformation plate and screened for DNAconstruct incorporation using Southern blots assays and expression ofthe thioesterase constructs were screened using RT-PCR.

Positive clones were picked from both the C12 and C14 thioesterasetransformation plates and screened for construct incorporation usingSouthern blot assays. Southern blot assays were carried out usingstandard methods (and described above in Example 3) using an optimized cprobes, based on the sequence in SEQ ID NO: 70 and SEQ ID NO: 71.Transforming plasmid DNA was run as a positive control. Out of theclones that were positive for construct incorporation, a subset wasselected for reverse transcription quantitative PCR (RT-qPCR) analysisfor C12 thioesterase and C14 thioesterase expression.

RNA isolation was performed using methods described in Example 3 aboveand RT-qPCR of the positive clones were performed using 20 ng of totalRNA from each clone using the below-described primer pair and iScriptSYBR Green RT-PCR kit (Bio-Rad Laboratories) according to manufacturer'sprotocol. Wildtype (non-transformed) Prototheca moriformis total RNA wasincluded as a negative control. mRNA expression was expressed asrelative fold expression (RFE) as compared to negative control. Theprimers that were used in the C12 thioesterase transformation RT-qPCRscreening were:

U. californica C12 thioesterase PCR primers:

(SEQ ID NO: 74) Forward: 5′ CTGGGCGACGGCTTCGGCAC 3′ (SEQ ID NO: 75)Reverse: 5′ AAGTCGCGGCGCATGCCGTT 3′

The primers that were used in the C14 thioesterase transformationRT-qPCR screening were:

Cinnamomum camphora C14 thioesterase PCR primers:

(SEQ ID NO: 76) Forward: 5′ TACCCCGCCTGGGGCGACAC 3′ (SEQ ID NO: 77)Reverse: 5′ CTTGCTCAGGCGGCGGGTGC 3′

RT-qPCR results for C12 thioesterase expression in the positive clonesshowed an increased RFE of about 40 fold to over 2000 fold increasedexpression as compared to negative control. Similar results were seenwith C14 thioesterase expression in the positive clones with an increaseRFE of about 60-fold to over 1200 fold increased expression as comparedto negative control.

A subset of the positive clones from each transformation (as screened bySouthern blotting and RT-qPCR assays) were selected and grown undernitrogen-replete conditions and analyzed for total lipid production andprofile. Lipid samples were prepared from dried biomass from each clone.20-40 mg of dried biomass from each transgenic clone was resuspended in2 mL of 3% H₂SO₄ in MeOH, and 200 ul of toluene containing anappropriate amount of a suitable internal standard (C19:0) was added.The mixture was sonicated briefly to disperse the biomass, then heatedat 65-70° C. for two hours. 2 mL of heptane was added to extract thefatty acid methyl esters, followed by addition of 2 mL of 6% K₂CO₃ (aq)to neutralize the acid. The mixture was agitated vigorously, and aportion of the upper layer was transferred to a vial containing Na₂SO₄(anhydrous) for gas chromatography analysis using standard FAME GC/FID(fatty acid methyl ester gas chromatography flame ionization detection)methods. Lipid profile (expressed as Area %) of the positive clones ascompared to wildtype negative control are summarized in Tables 15 and 16below. As shown in Table 15, the fold increase of C12 production in theC12 transformants ranged from about a 5-fold increase (clone C12-5) toover 11-fold increase (clone C12-1). Fold increase of C14 production inthe C14 transformants ranged from about a 1.5 fold increase to about a2.5 fold increase.

TABLE 15 Summary of total lipid profile of the Prototheca moriformis C12thioesterase transformants. Wildtype C12-1 C12-2 C12-3 C12-4 C12-5 C12-6C12-7 C12-8 C6:0 0.03 nd nd nd nd nd nd nd nd C8:0 0.11 0.09 nd 0.11 ndnd nd nd nd C10:0 nd nd nd 0.01 0.01 nd nd 0.01 nd C12:0 0.09 1.04 0.270.72 0.71 0.50 0.67 0.61 0.92 C14:0 2.77 2.68 2.84 2.68 2.65 2.79 2.732.56 2.69 C14:1 0.01 nd nd 0.02 nd nd nd 0.01 nd C15:0 0.30 0.09 0.100.54 0.19 0.09 0.13 0.97 0.09 C15:1 0.05 nd nd 0.02 nd nd nd nd nd C16:024.13 23.12 24.06 22.91 22.85 23.61 23.14 21.90 23.18 C16:1 0.57 0.620.10 0.52 0.69 0.63 0.69 0.49 0.63 C17:0 0.47 0.24 0.27 1.02 0.36 0.170.26 2.21 0.19 C17:1 0.08 nd 0.09 0.27 0.10 0.05 0.09 0.80 0.05 C18:0 ndnd 2.14 1.75 2.23 2.16 2.38 1.62 2.47 C18:1 22.10 23.15 24.61 21.9023.52 19.30 22.95 20.22 22.85 C18:1 nd 0.33 0.24 nd nd 0.09 0.09 nd 0.11C18:2 37.16 34.71 35.29 35.44 35.24 36.29 35.54 36.01 35.31 C18:3 11.6811.29 9.26 11.62 10.76 13.61 10.64 11.97 10.81 alpha C20:0 0.15 0.160.19 0.16 0.16 0.14 0.18 0.14 0.18 C20:1 0.22 0.17 0.19 0.20 0.21 0.190.21 0.20 0.21 C20:2 0.05 nd 0.04 0.05 0.05 0.05 0.04 0.05 0.04 C22:0 ndnd nd 0.01 nd nd nd 0.02 nd C22:1 nd nd nd nd nd 0.01 nd 0.01 nd C20:30.05 nd 0.07 0.06 0.06 0.10 0.07 0.05 0.06 C20:4 nd nd nd nd nd 0.02 ndnd nd C24:0 nd nd 0.24 0.01 0.20 0.19 0.19 0.14 0.20

TABLE 16 Summary of total lipid profile of the Prototheca moriformis C14thioesterase transformants. Wildtype C14-1 C14-2 C14-3 C14-4 C14-5 C14-6C14-7 C6:0 0.03 nd nd nd nd nd nd nd C8:0 0.11 nd nd nd nd nd nd ndC10:0 nd 0.01 nd 0.01 nd 0.01 nd nd C12:0 0.09 0.20 0.16 0.25 0.21 0.190.40 0.17 C14:0 2.77 4.31 4.76 4.94 4.66 4.30 6.75 4.02 C14:1 0.01 nd0.01 nd nd 0.01 nd nd C15:0 0.30 0.43 0.45 0.12 0.09 0.67 0.10 0.33C15:1 0.05 nd nd nd nd nd nd nd C16:0 24.13 22.85 23.20 23.83 23.8423.48 24.04 23.34 C16:1 0.57 0.65 0.61 0.60 0.60 0.47 0.56 0.67 C17:00.47 0.77 0.76 0.21 0.19 1.11 0.18 0.54 C17:1 0.08 0.23 0.15 0.06 0.050.24 0.05 0.12 C18:0 nd 1.96 1.46 2.48 2.34 1.84 2.50 2.06 C18:1 22.1022.25 19.92 22.36 20.57 19.50 20.63 22.03 C18:1 nd nd nd nd nd nd 0.10nd C18:2 37.16 34.97 36.11 34.35 35.70 35.49 34.03 35.60 C18:3 11.6810.71 12.00 10.15 11.03 12.08 9.98 10.47 alpha C20:0 0.15 0.16 0.19 0.170.17 0.14 0.18 0.16 C20:1 0.22 0.20 0.12 .019 0.19 0.19 0.17 0.20 C20:20.05 0.04 0.02 0.03 0.04 0.05 0.03 0.04 C22:0 nd nd nd nd 0.02 0.01 ndnd C22:1 nd 0.01 nd nd nd nd nd 0.01 C20:3 0.05 0.08 0.03 0.06 0.09 0.050.05 0.07 C20:4 nd 0.01 nd nd nd nd 0.02 nd C24:0 nd 0.17 0.14 0.19 0.200.16 0.22 0.17

The above-described experiments indicate the successful transformationof Prototheca moriformis (UTEX 1435) with transgene constructs of twodifferent thioesterases (C12 and C14), which involved not only thesuccessful expression of the transgene, but also the correct targetingof the expressed protein to the plastid and a functional effect (theexpected change in lipid profile) as a result of the transformation. Thesame transformation experiment was performed using an expressionconstruct containing a codon-optimized (according to Table 1) Cupheahookeriana C8-10 thioesterase coding region with the native plastidtargeting sequence (SEQ ID NO: 78) yielded no change in lipid profile.While the introduction of the Cuphea hookeriana C8-10 transgene intoPrototheca moriformis (UTEX 1435) was successful and confirmed bySouthern blot analysis, no change in C8 or C10 fatty acid production wasdetected in the transformants compared to the wildtype strain.

Example 5 Generation of Prototheca moriformis Strain with ExogenousPlant TE with Algal Plastid Targeting Sequence

In order to investigate whether the use of algal chloroplast/plastidtargeting sequences would improve medium chain (C8-C14) thioesteraseexpression and subsequent medium chain lipid production in Protothecamoriformis (UTEX 1435), several putative algal plastid targetingsequences were cloned from Chlorella protothecoides and Protothecamoriformis. Thioesterase constructs based on Cuphea hookeriana C8-10thioesterase, Umbellularia californica C12 thioesterase, and Cinnamomumcamphora C14 thioesterase were made using made with a Chlorellasorokiniana glutamate dehydrogenase promoter/5′UTR and a Chlorellavulgaris nitrate reductase 3′UTR. The thioesterase coding sequences weremodified by removing the native plastid targeting sequences andreplacing them with plastid targeting sequences from the Chlorellaprotothecoides and the Prototheca moriformis genomes. The thioesteraseexpression constructs and their corresponding sequence identificationnumbers are listed below. Each transformation plasmid also contained aNeo resistance construct that was identical to the ones described inExample 3 above. Additionally, another algal-derived promoter, theChlamydomonas reinhardtii β-tubulin promoter, was also tested inconjunction with the thioesterase constructs. “Native” plastid targetingsequence refers to the higher plant thioesterase plastid targetingsequence. A summary of the constructs used in these experiments isprovided below:

Construct Promoter/ Plastid Name 5′UTR targeting seq Gene 3′UTR SEQ IDNO. Construct 1 C. sorokiniana C. protothecoides Cuphea C. vulgaris SEQID NO: 79 glutamate stearoyl ACP hookeriana nitrate dehydrogenasedesaturase C8-10 TE reductase Construct 2 C. sorokiniana P. moriformisCuphea C. vulgaris SEQ ID NO: 80 glutamate delta 12 fatty hookeriananitrate dehydrogenase acid desaturase C8-10 TE reductase Construct 3 C.sorokiniana P. moriformis Cuphea C. vulgaris SEQ ID NO: 81 glutamateisopentenyl hookeriana nitrate dehydrogenase diphosphate C8-10 TEreductase synthase Construct 4 C. sorokiniana P. moriformis UmbellulariaC. vulgaris SEQ ID NO: 82 glutamate isopentenyl californica nitratedehydrogenase diphosphate C12 TE reductase synthase Construct 5 C.sorokiniana P. moriformis Umbellularia C. vulgaris SEQ ID NO: 83glutamate stearoyl ACP californica nitrate dehydrogenase desaturase C12TE reductase Construct 6 C. sorokiniana C. protothecoides UmbellulariaC. vulgaris SEQ ID NO: 84 glutamate stearoyl ACP californica nitratedehydrogenase desaturase C12 TE reductase Construct 7 C. sorokiniana P.moriformis Umbellularia C. vulgaris SEQ ID NO: 85 glutamate delta 12fatty californica nitrate dehydrogenase acid desaturase C12 TE reductaseConstruct 8 C. sorokiniana C. protothecoides Cinnamomum C. vulgaris SEQID NO: 86 glutamate stearoyl ACP camphora nitrate dehydrogenasedesaturase C14 TE reductase Construct 9 Chlamydomonas Native Cuphea C.vulgaris SEQ ID NO: 113 reinhardtii hookeriana nitrate β-tubulin C8-10TE reductase Construct 10 Chlamydomonas P. moriformis Cuphea C. vulgarisSEQ ID NO: 114 reinhardtii isopentenyl hookeriana nitrate β-tubulindiphosphate C8-10 TE reductase synthase Construct 11 Chlamydomonas P.moriformis Cuphea C. vulgaris SEQ ID NO: 115 reinhardtii delta 12 fattyhookeriana nitrate β-tubulin acid desaturase C8-10 TE reductaseConstruct 12 Chlamydomonas C. protothecoides Cuphea C. vulgaris SEQ IDNO: 116 reinhardtii stearoyl ACP hookeriana nitrate β-tubulin desaturaseC8-10 TE reductase Construct 13 Chlamydomonas P. moriformis Cuphea C.vulgaris SEQ ID NO: 117 reinhardtii stearoyl ACP hookeriana nitrateβ-tubulin desaturase C8-10 TE reductase Construct 14 ChlamydomonasNative Umbellularia C. vulgaris SEQ ID NO: 118 reinhardtii californicanitrate β-tubulin C12 TE reductase Construct 15 Chlamydomonas P.moriformis Umbellularia C. vulgaris SEQ ID NO: 119 reinhardtiiisopentenyl californica nitrate β-tubulin diphosphate C12 TE reductaseConstruct 16 Chlamydomonas P. moriformis Umbellularia C. vulgaris SEQ IDNO: 120 reinhardtii delta 12 fatty californica nitrate β-tubulin aciddesaturase C12 TE reductase Construct 17 Chlamydomonas C. protothecoidesUmbellularia C. vulgaris SEQ ID NO: 121 reinhardtii stearoyl ACPcalifornica nitrate β-tubulin desaturase C12 TE reductase Construct 18Chlamydomonas P. moriformis Umbellularia C. vulgaris SEQ ID NO: 122reinhardtii stearoyl ACP californica nitrate β-tubulin desaturase C12 TEreductase Construct 19 Chlamydomonas Native Cinnamomum C. vulgaris SEQID NO: 123 reinhardtii camphora nitrate β-tubulin C14 TE reductaseConstruct 20 Chlamydomonas P. moriformis Cinnamomum C. vulgaris SEQ IDNO: 124 reinhardtii isopentenyl camphora nitrate β-tubulin diphosphateC14 TE reductase synthase Construct 21 Chlamydomonas P. moriformisCinnamomum C. vulgaris SEQ ID NO: reinhardtii delta 12 fatty camphoranitrate β-tubulin acid desaturase C14 TE reductase Construct 22Chlamydomonas C. protothecoides Cinnamomum C. vulgaris SEQ ID NO: 87reinhardtii stearoyl ACP camphora nitrate β-tubulin desaturase C14 TEreductase Construct 23 Chlamydomonas P. moriformis Cinnamomum C.vulgaris SEQ ID NO: 88 reinhardtii stearoyl ACP camphora nitrateβ-tubulin desaturase C14 TE reductase

Each construct was transformed into Prototheca moriformis (UTEX 1435)and selection was performed using G418 using the methods described inExample 4 above. Several positive clones from each transformation werepicked and screened for the presence thioesterase transgene usingSouthern blotting analysis. Expression of the thioesterase transgene wasconfirmed using RT-PCR. A subset of the positive clones (as confirmed bySouthern blotting analysis and RT-PCR) from each transformation wasselected and grown for lipid profile analysis. Lipid samples wereprepared from dried biomass samples of each clone and lipid profileanalysis was performed using acid hydrolysis methods described inExample 4. Changes in area percent of the fatty acid corresponding tothe thioesterase transgene were compared to wildtype levels, and clonestransformed with a thioesterase with the native plastid targetingsequence.

As mentioned in Example 4, the clones transformed with Cuphea hookerianaC8-10 thioesterase constructs with the native plastid targeting sequencehad the same level of C8 and C10 fatty acids as wildtype. The clonestransformed with Cuphea hookeriana C8-10 thioesterase constructs(Constructs 1-3) with algal plastid targeting sequences had over a10-fold increase in C10 fatty acids for Construct 3 and over 40-foldincrease in C10 fatty acids for Constructs 1 and 2 (as compared towildtype). The clones transformed with Umbellularia californica C12thioesterase constructs with the native plastid targeting sequence had amodest 6-8 fold increase in C12 fatty acid levels as compared towildtype. The clones transformed with the Umbellularia californica C12thioesterase constructs with the algal plasid targeting constructs(Constructs 4-7) had over an 80-fold increase in C12 fatty acid levelfor Construct 4, about an 20-fold increase in C12 fatty acid level forConstruct 6, about a 10-fold increase in C12 fatty acid level forConstruct 7 and about a 3-fold increase in C12 fatty acid level forConstruct 5 (all compared to wildtype). The clones transformed withCinnamomum camphora C14 thioesterase with either the native plastidtargeting sequence or the construct 8 (with the Chlorella protothecoidesstearoyl ACP desaturase plastid targeting sequence) had about a 2-3 foldincrease in C14 fatty acid levels as compared to wildtype. In generalclones transformed with an algal plastid targeting sequence thioesteraseconstructs had a higher fold increase in the corresponding chain-lengthfatty acid levels than when using the native higher plant targetingsequence.

A. Chlamydomonas reinhardtii β-Tubulin Promoter

Additional heterologous thioesterase expression constructs were preparedusing the Chlamydomonas reinhardtii β-tubulin promoter instead of the C.sorokinana glutamate dehydrogenase promoter. The construct elements andsequence of the expression constructs are listed above. Each constructwas transformed into Prototheca moriformis UTEX 1435 host cells usingthe methods described above. Lipid profiles were generated from a subsetof positive clones for each construct in order to assess the success andproductivity of each construct. The lipid profiles compare the fattyacid levels (expressed in area %) to wildtype host cells. The “Mean”column represents the numerical average of the subset of positiveclones. The “Sample” column represents the best positive clone that wasscreened (best being defined as the sample that produced the greatestchange in area % of the corresponding chain-length fatty acidproduction). The “low-high” column represents the lowest area % and thehighest area % of the fatty acid from the clones that were screened. Thelipid profiles results of Constructs 9-23 are summarized below.

Fatty Acid wildtype Mean Sample low/high Construct 9. Cuphea hookerianaC8-10 TE C 8:0 0 0.05 0.30   0-0.29 C 10:0 0.01 0.63 2.19   0-2.19 C12:0 0.03 0.06 0.10   0-0.10 C 14:0 1.40 1.50 1.41 1.36-3.59 C 16:024.01 24.96 24.20 C 16:1 0.67 0.80 0.85 C 17:0 0 0.16 0.16 C 17:1 0 0.910 C 18:0 4.15 17.52 3.19 C 18:1 55.83 44.81 57.54 C 18:2 10.14 7.58 8.83C 18:3α 0.93 0.68 0.76 C 20:0 0.33 0.21 0.29 C 24:0 0 0.05 0.11Construct 10. Cuphea hookeriana C8-10 TE C 8:0 0 0.01 0.02   0-0.03 C10:0 0 0.16 0.35   0-0.35 C 12:0 0.04 0.05 0.07   0-0.07 C 14:0 1.131.62 1.81   0-0.05 C 14:1 0 0.04 0.04 C 15:0 0.06 0.05 0.05 C 16:0 19.9426.42 28.08 C 16:1 0.84 0.96 0.96 C 17:0 0.19 0.14 0.13 C 17:1 0.10 0.060.05 C 18:0 2.68 3.62 3.43 C 18:1 63.96 54.90 53.91 C 18:2 9.62 9.839.11 C18:3γ 0 0.01 0 C 18:3α 0.63 0.79 0.73 C 20:0 0.26 0.35 0.33 C 20:10.06 0.08 0.09 C 20:1 0.08 0.06 0.07 C 22:0 0 0.08 0.09 C 24:0 0.13 0.130.11 Construct 11. Cuphea hookeriana C8-10 TE C 8:0 0 0.82 1.57   0-1.87C 10:0 0 3.86 6.76   0-6.76 C 12:0 0.04 0.13 0.20 0.03-0.20 C 14:0 1.131.80 1.98 1.64-2.05 C 14:1 0 0.04 0.04 C 15:0 0.06 0.06 0.06 C 16:019.94 25.60 25.44 C 16:1 0.84 1.01 1.02 C 17:0 0.19 0.13 0.11 C 17:10.10 0.06 0.05 C 18:0 2.68 2.98 2.38 C 18:1 63.96 51.59 48.85 C 18:29.62 9.85 9.62 C18:3γ 0 0.01 0 C 18:3α 0.63 0.91 0.92 C 20:0 0.26 0.290.26 C 20:1 0.06 0.06 0 C 20:1 0.08 0.06 0.03 C 22:0 0 0.08 0.08 C 24:00.13 0.06 0 Construct 12. Cuphea hookeriana C8-10 TE C 8:0 0 0.31 0.85  0-0.85 C 10:0 0 2.16 4.35 0.20-4.35 C 12:0 0.04 0.10 0.15   0-0.18 C14:0 1.13 1.96 1.82 1.66-2.97 C 14:1 0 0.03 0.04 C 15:0 0.06 0.07 0.07 C16:0 19.94 26.08 25.00 C 16:1 0.84 1.04 0.88 C 17:0 0.19 0.16 0.16 C17:1 0.10 0.05 0.07 C 18:0 2.68 3.02 3.19 C 18:1 63.96 51.08 52.15 C18:2 9.62 11.44 9.47 C18:3γ 0 0.01 0 C 18:3α 0.63 0.98 0.90 C 20:0 0.260.30 0.28 C 20:1 0.06 0.06 0.05 C 20:1 0.08 0.04 0 C 22:0 0 0.07 0 C24:0 0.13 0.05 0 Construct 14. Umbellularia californica C12 TE C 10:00.01 0.02 0.03 0.02-0.03 C 12:0 0.03 2.62 3.91 0.04-3.91 C 14:0 1.401.99 2.11 1.83-2.19 C 16:0 24.01 27.64 27.01 C 16:1 0.67 0.92 0.92 C18:0 4.15 2.99 2.87 C 18:1 55.83 53.22 52.89 C 18:2 10.14 8.68 8.41 C18:3α 0.93 0.78 0.74 C 20:0 0.33 0.29 0.27 Construct 15. Umbellulariacalifornica C12 TE C 10:0 0 0.05 0.08   0-0.08 C 12:0 0.04 8.12 12.80 4.35-12.80 C 13:0 0 0.02 0.03   0-0.03 C 14:0 1.13 2.67 3.02 2.18-3.37C 14:1 0 0.04 0.03 0.03-0.10 C 15:0 0.06 0.07 0.06 C 16:0 19.94 25.2623.15 C 16:1 0.84 0.99 0.86 C 17:0 0.19 0.14 0.14 C 17:1 0.10 0.05 0.05C 18:0 2.68 2.59 2.84 C 18:1 63.96 46.91 44.93 C 18:2 9.62 10.59 10.01 C18:3α 0.63 0.92 0.83 C 20:0 0.26 0.27 0.24 C 20:1 0.06 0.06 0.06 C 20:10.08 0.05 0.04 C 22:0 0 0.07 0.09 C 24:0 0.13 0.13 0.12 Construct 16.Umbellularia californica C12 TE C 10:0 0 0.03 0.04 0.02-0.04 C 12:0 0.042.43 5.32 0.98-5.32 C 13:0 0 0.01 0.02   0-0.02 C 14:0 1.13 1.77 1.931.62-1.93 C 14:1 0 0.03 0.02 0.02-0.04 C 15:0 0.06 0.06 0.05 C 16:019.94 24.89 22.29 C 16:1 0.84 0.91 0.82 C 17:0 0.19 0.16 0.15 C 17:10.10 0.06 0.06 C 18:0 2.68 3.81 3.67 C 18:1 63.96 53.19 52.82 C 18:29.62 10.38 10.57 C 18:3α 0.63 0.80 0.77 C 20:0 0.26 0.35 0.32 C 20:10.06 0.06 0.07 C 20:1 0.08 0.07 0.08 C 22:0 0 0.08 0.07 C 24:0 0.13 0.150.14 Construct 17. Umbellularia californica C12 TE C 10:0 0 0.04 0.070.03-0.08 C 12:0 0.04 7.02 14.11  4.32-14.11 C 13:0 0 0.03 0.040.01-0.04 C 14:0 1.13 2.25 3.01 1.95-3.01 C 14:1 0 0.03 0.03 0.02-0.03 C15:0 0.06 0.06 0.06 C 16:0 19.94 23.20 21.46 C 16:1 0.84 0.82 0.77 C17:0 0.19 0.15 0.14 C 17:1 0.10 0.06 0.06 C 18:0 2.68 3.47 2.93 C 18:163.96 50.30 45.17 C 18:2 9.62 10.33 9.98 C18:3γ 0 0.01 0 C 18:3α 0.630.84 0.86 C 20:0 0.26 0.32 0.27 C 20:1 0.06 0.07 0.06 C 20:1 0.08 0.060.06 C 22:0 0 0.08 0.09 C 24:0 0.13 0.14 0.13 Construct 18. Umbellulariacalifornica C12 TE C 10:0 0 0.03 0.05 0.01-0.05 C 12:0 0.04 5.06 7.770.37-7.77 C 13:0 0 0.02 0   0-0.03 C 14:0 1.13 2.11 2.39 1.82-2.39 C14:1 0 0.03 0.03 0.02-0.05 C 15:0 0.06 0.06 0.06 C 16:0 19.94 24.6023.95 C 16:1 0.84 0.86 0.83 C 17:0 0.19 0.15 0.14 C 17:1 0.10 0.06 0.05C 18:0 2.68 3.31 2.96 C 18:1 63.96 51.26 49.70 C 18:2 9.62 10.18 10.02C18:3γ 0 0.01 0.02 C 18:3α 0.63 0.86 0.86 C 20:0 0.26 0.32 0.29 C 20:10.06 0.05 0.05 C 20:1 0.08 0.07 0.04 C 22:0 0 0.08 0.08 C 24:0 0.13 0.130.13 Construct 19. Cinnamomum camphora C14 TE C 10:0 0.02 0.01 0.010.01-0.02 C 12:0 0.05 0.27 0.40 0.08-0.41 C 14:0 1.52 4.47 5.812.10-5.81 C 16:0 25.16 28.14 28.55 C 16:1 0.72 0.84 0.82 C 18:0 3.703.17 2.87 C 18:1 54.28 51.89 51.01 C 18:2 12.24 9.36 8.62 C 18:3α 0.870.74 0.75 C 20:0 0.33 0.33 0.31 Construct 20. Cinnamomum camphora C14 TEC 10:0 0.01 0.01 0.02 0.01-0.02 C 12:0 0.03 0.39 0.65 0.08-0.65 C 13:0 00.01 0.01 0.01-0.02 C 14:0 1.40 5.61 8.4 2.1-8.4 C 14:1 0 0.03 0.030.02-0.03 C 15:0 0 0.06 0.07 C 16:0 24.01 25.93 25.57 C 16:1 0.67 0.750.71 C 17:0 0 0.13 0.12 C 17:1 0 0.05 0.05 C 18:0 4.15 3.30 3.23 C 18:155.83 51.00 48.48 C 18:2 10.14 10.38 10.35 C 18:3α 0.93 0.91 0.88 C 20:00.33 0.35 0.32 C 20:1 0 0.08 0.08 C 20:1 0 0.07 0.07 C 22:0 0 0.08 0.08C 24:0 0 0.14 0.13 Construct 21. Cinnamomum camphora C14 TE C 10:0 0.010.01 0.01   0-0.01 C 12:0 0.03 0.10 0.27 0.04-0.27 C 14:0 1.40 2.28 4.401.47-4.40 C 16:0 24.01 26.10 26.38 C 16:1 0.67 0.79 0.73 C 17:0 0 0.150.16 C 17:1 0 0.06 0.06 C 18:0 4.15 3.59 3.51 C 18:1 55.83 53.53 50.86 C18:2 10.14 10.83 11.11 C 18:3α 0.93 0.97 0.87 C 20:0 0.33 0.36 0.37 C20:1 0 0.09 0.08 C 20:1 0 0.07 0.07 C 22:0 0 0.09 0.09 Construct 22.Cinnamomum camphora C14 TE C 10:0 0.01 0.02 0.02 0.02-0.02 C 12:0 0.031.22 1.83 0.59-1.83 C 13:0 0 0.02 0.03 0.01-0.03 C 14:0 1.40 12.77 17.33 7.97-17.33 C 14:1 0 0.02 0.02 0.02-0.04 C 15:0 0 0.07 0.08 C 16:0 24.0124.79 24.22 C 16:1 0.67 0.64 0.58 C 17:0 0 0.11 0.10 C 17:1 0 0.04 0.04C 18:0 4.15 2.85 2.75 C 18:1 55.83 45.16 41.23 C 18:2 10.14 9.96 9.65 C18:3α 0.93 0.91 0.85 C 20:0 0.33 0.30 0.30 C 20:1 0 0.07 0.06 C 20:1 00.06 0.05 C 22:0 0 0.08 0.08 Construct 23. Cinnamomum camphora C14 TE C10:0 0.01 0.01 0.02   0-0.02 C 12:0 0.05 0.57 1.08 0.16-1.08 C 13:0 00.02 0.02   0-0.02 C 14:0 1.45 7.18 11.24  2.96-11.24 C 14:1 0.02 0.030.03 0.02-0.03 C 15:0 0.06 0.07 0.07 C 16:0 24.13 25.78 25.21 C 16:10.77 0.72 0.66 C 17:0 0.19 0.13 0.11 C 17:1 0.08 0.05 0.04 C 18:0 3.533.35 3.12 C 18:1 56.15 49.65 46.35 C 18:2 11.26 10.17 9.72 C 18:3α 0.840.95 0.83 C 20:0 0.32 0.34 0.32 C 20:1 0.09 0.08 0.09 C 20:1 0.07 0.050.06 C 22:0 0.07 0.08 0.08 C 24:0 0.13 0.13 0.12

Constructs 9-13 were expression vectors containing the Cuphea hookerianaC8-10 thioesterase construct. As can be seen in the data summariesabove, the best results were seen with Construct 11, with the Sample C8fatty acid being 1.57 Area % (as compared to 0 in wildtype) and C10fatty acid being 6.76 Area % (as compared to 0 in wildtype). There wasalso a modest increase in C12 fatty acids (approximately 2-5 foldincrease). While the native plastid targeting sequence produced nochange when under the control of the C. sorokinana glutamatedehydrogenase promoter, the same expression construct driven by the C.reinhardtii β-tubulin promoter produced significant changes in C8-10fatty acids in the host cell. This is further evidence of theidiosyncrasies of heterologous expression of thioesterases in Protothecaspecies. All of the clones containing the C. reinhardtii β-tubulinpromoter C8-10 thioesterase construct had greater increases in C8-10fatty acids than the clones containing the C. sorokinana glutamatedehydrogenase promoter C8-10 thioesterase construct. Lipid profile datafor Construct 13 was not obtained and therefore, not included above.

Constructs 14-18 were expression vectors containing the Umbellulariacalifornica C12 thioesterase construct. As can be seen in the datasummaries above, the best results were seen with Constructs 15 (P.moriformis isopentenyl diphosphate synthase plastid targeting sequence)and 17 (C. protothecoides stearoyl ACP desaturase plastid targetingsequence). The greatest change in C12 fatty acid production was seenwith Construct 17, with C12 fatty acids levels of up to 14.11 area %, ascompared to 0.04 area % in wildtype. Modest changes (about 2-fold) werealso seen with C14 fatty acid levels. When compared to the sameconstructs with the C. sorokinana glutamate dehydrogenase promoter, thesame trends were true with the C. reinhardtii β-tubulin promoter—the C.protothecoides stearoyl ACP desaturase and P. moriformis isopentenyldiphosphate synthase plastid targeting sequences produced the greatestchange in C12 fatty acid levels with both promoters.

Constructs 19-23 were expression vectors containing the Cinnamomumcamphora C14 thioesterase construct. As can be seen in the datasummaries above, the best results were seen with Constructs 22 andConstruct 23. The greatest change in C14 fatty acid production was seenwith Construct 22, with C14 fatty acid levels of up to 17.35 area %(when the values for C140 and C141 are combined), as compared to 1.40%in wildtype. Changes in C12 fatty acids were also seen (5-60 fold). Whencompared to the same constructs with the C. sorokinana glutamatedehydrogenase promoter, the same trends were true with the C.reinhardtii β-tubulin promoter—the C. protothecoides stearoyl ACPdesaturase and P. moriformis stearoyl ACP desaturase plastid targetingsequences produced the greatest change in C14 fatty acid levels withboth promoters. Consistently with all thioesterase expressionconstructs, the C. reinhardtii β-tubulin promoter constructs producedgreater changes in C8-14 fatty acid levels than the C. sorokinianaglutamate dehydrogenase

Two positive clones from the Construct 22 were selected and grown underhigh selective pressure (50 mg/L G418). After 6 days in culture, theclones were harvested and their lipid profile was determined using themethods described above. The lipid profile data is summarized below andis expressed in area %.

Construct 22 clones + 50 mg/L G418 Construct Construct Fatty Acid 22 A22 B C 12:0 3.21 3.37 C 14:0 27.55 26.99 C 16:0 25.68 24.37 C 16:1 0.990.92 C 18:0 1.37 1.23 C 18:1 28.35 31.07 C 18:2 11.73 11.05 C 18:3α 0.920.81 C 20:0 0.16 0.17

Both clones, when grown under constant, high selective pressure,produced an increased amount of C14 and C12 fatty acids, about doublethe levels seen with Construct 22 above. These clones yielded over 30area % of C12-14 fatty acids, as compared to 1.5 area % of C12-14 fattyacids seen in wildtype cells.

Example 6 Heterologous Expression of Cuphea palustris and Ulmusamericanca Thioesterase in Prototheca

Given the success of the above-described heterologous expressionthioesterases in Prototheca species, expression cassettes containingcodon-optimized (according to Table 1) sequences encoding fatty acyl-ACPthioesterases from Cuphea palustris and Ulmus americana were constructedand described below.

Construct Promoter/ Plastid Name 5′UTR targeting seq Gene 3′UTR SEQ IDNO. Construct 27 C. reinhardtii C. protothecoides Cuphea C. vulgaris SEQID NO: 107 β-tubulin stearoyl ACP palustris nitrate desaturasethioesterase reductase

The Ulmus americana (codon-optimized coding sequence) can be insertedinto the expression cassette. The codon-optimized coding sequencewithout the native plastid targeting sequence for the Ulmus americanathioesterase is listed as SEQ ID NO: 108 and can be fused any desiredplastid targeting sequence and expression element (i.e., promoter/5′UTRand 3′UTR).

These expression cassettes can be transformed in to Prototheca speciesusing the methods described above. Positive clones can be screened withthe inclusion of an antibiotic resistance gene (e.g, neoR) on theexpression construct and screened on G418-containing plates/media.Positive clones can be confirmed using Southern blot assays with probesspecific to the heterologous thioesterase coding region and expressionof the construct can also be confirmed using RT-PCR and primers specificto the coding region of the heterologous thioesterase. Secondaryconfirmation of positive clones can be achieved by looking for changesin levels of fatty acids in the host cell's lipid profile. As seen inthe above Examples, heterologous expression in Prototheca species ofthioesterase can be idiosyncratic to the particular thioesterase.Promoter elements and plastid targeting sequences (and other expressionregulatory elements) can be interchanged until the expression of thethioesterase (and the subsequent increase in the corresponding fattyacid) reaches a desired level.

Example 7 Dual Transformants—Simultaneous Expression of Two HeterologousProteins

Microalgae strain Prototheca moriformis (UTEX 1435) was transformedusing the above disclosed methods with a expression construct containingthe yeast sucrose invertase suc2 gene encoding the secreted form of theS. cerevisiae invertase. Successful expression of this gene andtargeting to the periplasm results in the host cell's ability to grow on(and utilize) sucrose as a sole carbon source in heterotrophicconditions (as demonstrated in Example 3 above). The second set of genesexpressed are thioesterases which are responsible for the cleavage ofthe acyl moiety from the acyl carrier protein. Specifically,thioesterases from Cuphea hookeriana (a C8-10 preferring thioesterase),Umbellularia californica (a C12 preferring thioesterase), and Cinnamomumcamphora (a C14 preferring thioesterase). These thioesterase expressioncassettes were cloned as fusions with N-terminal microalgal plastidtargeting sequences from either Prototheca moriformis or Chlorellaprotothecoides, which have been shown (in the above Examples) to be moreoptimal than the native higher plant plastid targeting sequences. Thesuccessful expression of the thioesterase genes and the targeting to theplastid resulted in measurable changes in the fatty acid profiles withinthe host cell. These changes in profiles are consistent with theenzymatic specificity or preference of each thioesterase. Below is asummary of dual expression contructs that were assembled and transformedinto Prototheca moriformis (UTEX 1435). Each construct contained theyeast suc2 gene under the control of the C. reinhardtii β-tubulin5′UTR/promoter and contained the C. vulgaris nitrate reductase 3′UTR anda higher plant thioesterase with a microalgal plastid targeting sequencereplacing the native sequence under the control of C. sorokinanaglutamate dehydrogenase 5′UTR and contained the C. vulgaris nitratereductase 3′UTR. Below is a summary of the thioesterase portion of theconstructs that were assembled and transformed into Protothecamoriformis (UTEX 1435). The entire dual expression cassette with thesuc2 gene and the thioesterase gene and the is listed in the SequenceIdentification Listing.

Construct Promoter/ Plastid Name 5′UTR targeting seq Gene 3′UTR SEQ IDNO. Construct 24 C. sorokiniana C. protothecoides Cuphea C. vulgaris SEQID NO: 109 glutamate stearoyl ACP hookeriana nitrate dehydrogenasedesaturase C8-10 TE reductase Construct 25 C. sorokinana P. moriformisUmbellularia C. vulgaris SEQ ID NO: 110 glutamate isopentenylcalifornica nitrate dehydrogenase diphosphate C12 TE reductase synthaseConstruct 26 C. sorokinana C. protothecoides Cinnamomum C. vulgaris SEQID NO: 111 glutamate stearoyl ACP camphora nitrate dehydrogenasedesaturase C14 TE reductaseSimilar dual expression constructs with the thioesterase cassettesdescribed in Example 5 (e.g., under the control of a different promotersuch as C. reinhardtiiβ-tubulin promoter/5′UTR) can also be generatedusing standard molecular biology methods and methods described herein.

Positive clones containing each of expression constructs were screenedusing their ability to grow on sucrose-containing plates, where sucroseis the sole-carbon source, as the selection factor. A subset of thesepositive clones from each construct transformation was selected and thepresence of the expression construct was confirmed using Southern blotassays. The function of the yeast sucrose invertase was also confirmedusing a sucrose hydrolysis assay. Positive clones were selected andgrown in media containing sucrose as the sole carbon source at astarting concentration of 40 g/L. A negative control of wildtypePrototheca moriformis (UTEX 1435) grown in media containing glucose asthe sole carbon source at the same 40 g/L starting concentration wasalso included. Utilization of sucrose was measured throughout the courseof the experiment by measuring the level of sucrose in the media using aYSI 2700 Biochemistry Analyzer with a sucrose-specific membrane. Aftersix days in culture, the cultures were harvested and processed for lipidprofile using the same methods as described above. The lipid profileresults are summarized below in Table 17 and are show in area %.

TABLE 17 Lipid profiles of dual transformants with suc2 sucroseinvertase and thioesterase. Fatty C24 C24 C24 C25 C25 C25 C26 C26 C26Acid Wt A B C A B C A B C C 10:0 0.01 0.03 0.04 0.08 0.01 0.01 0.01 0.010.01 0.0 C 12:0 0.04 0.04 0.04 0.04 0.28 0.40 0.10 0.04 0.04 0.13 C 14:01.6 1.55 1.53 1.56 1.59 1.59 1.60 1.65 1.56 2.69 C 14:1 0.03 0.03 0.030.02 0.03 0.03 0.03 0.03 0.03 0.03 C 15:0 0.04 0.03 0.03 0.04 0.04 0.030.03 0.03 0.03 0.04 C 16:0 29.2 29.1 29.0 28.6 28.9 28.6 29.0 28.8 29.527.5 C 16:1 0.86 0.80 0.79 0.82 0.77 0.81 0.82 0.79 0.79 0.86 C 17:0 0.10.08 0.08 0.09 0.09 0.08 0.09 0.08 0.08 0.09 C 17:1 0.04 0.03 0.03 0.040.03 0.03 0.03 0.03 0.03 0.04 C 18:0 3.26 3.33 3.37 3.27 3.36 3.28 3.183.33 3.36 3.03 C 18:1 54.5 53.9 54.1 53.9 53.5 53.7 53.5 54.2 53.9 52.7C 18:2 8.72 9.35 9.22 9.45 9.68 9.65 9.87 9.31 9.06 10.8 C 18:3 0.630.71 0.69 0.73 0.74 0.73 0.75 0.71 0.66 0.83 alpha C 20:0 0.29 0.31 0.310.31 0.32 0.32 0.31 0.32 0.31 0.29

All of the positive clones selected for the sucrose utilization assaywere able to hydrolyze the sucrose in the media and at the end of the 6day culture period, there were no measurable levels of sucrose in themedia. This data, in addition to the successful use of sucrose as aselection tool for positive clones, indicates that the exogenous yeastsuc2 sucrose invertase gene was targeted correctly and expressed in thetransformants. As show in Table 17 above, the clones expressingConstruct 24 (C8-10 thioesterase) had a measurable increase in C10 fattyacids (as high as an eight-fold increase). Likewise there weremeasurable increases in clones expressing Construct 25 (C12thioesterase) and Construct 26 (C14 thioesterase) in the correspondingmedium chain fatty acids. Taken together, the data shows the successfulsimultaneous expression in Prototheca moriformis two recombinantproteins (e.g., sucrose invertase and a fatty acid acyl-ACPthioesterase), both of which confer useful and quantifiable phenotypicchanges on the host organism.

Example 8 Effects of Glycerol on C10-C14 Fatty Acid Production in C14Thioesterase Transformants

Clones from all the thioesterase transformations were selected andfurther evaluated. One clone expressing Construct 8 (Cinnamomum camphoraC14 TE) was grown heterotrophically using different carbon sources:glucose only, fructose only and glycerol only. The glucose onlycondition resulted in higher cell growth and total lipid production whencompared to the fructose only and glycerol only conditions. However, theproportion of C12-14 fatty acids produced in the glycerol only conditionwas two-fold higher than that attained in the glucose only condition.

Example 9 Expression of Arabidopsis thaliana Invertase in Protohecamoriformis

Microalgae strain Prototheca moriformis (UTEX 1435) was transformedusing methods described above, with an expression construct containing acodon-optimized (according to Table 1) cell wall associated invertasefrom Arabidopsis thaliana. The Arabidoposis invertase sequence wasmodified to include the N-terminal 39 amino acids from yeast invertase(SUC2 protein) to ensure efficient targeting to the ER and ultimatelythe periplasm. To aid detection, a Flag epitope was added to theC-terminus of the recombinant protein. The transgene was cloned into anexpression vector with a Chlorella sorokinianna glutamate dehydrogenasepromoter/5′UTR region and a Chlorella vulgaris nitrate reductase 3′UTRregion. The DNA sequence of this transgene cassette is listed as SEQ IDNO: 89 and the translated amino acid sequence is listed as SEQ ID NO:90. Positive clones were screened and selected using sucrose-containingmedia/plates. A subset of the positive clones were confirmed for thepresence of the transgene and expression of invertase using Southernblot analysis and Western blot analysis for the Flag-tagged invertase.From these screens, 10 positive clones were chosen for lipidproductivity and sucrose utilization assays. All 10 clones were grown onmedia containing sucrose as the sole carbon source and a positivecontrol suc2 invertase transformant was also included. The negativecontrol, wildtype Prototheca moriformis, was also grown but on glucosecontaining media. After six days, the cells were harvested and dried andthe total percent lipid by dry cell weight was determined. The media wasalso analyzed for total sucrose consumption.

All ten positive clones were able to hydrolyze sucrose, however, mostclones grew about half as well as either wildtype or the positivecontrol suc2 yeast invertase transformant as determined by dry cellweight at the end of the experiment. Similarly, all ten positive clonesproduced about half as much total lipid when compared to wildtype or thepositive control transformant. This data demonstrate the successfulheterologous expression of diverse sucrose invertases in Prototheca.

Example 10 Heterologous Expression of Yeast Invertase (Suc2) inPrototheca krugani, Prototheca stagnora and Prototheca zopfii

To test the general applicability of the transformation methods for usein species of the genus Prototheca, three other Prototheca species wereselected: Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442)and Prototheca zopfii (UTEX 1438). These three strains were grown in themedia and conditions described in Example 1 and their lipid profileswere determined using the above described methods. A summary of thelipid profiles from the three Prototheca strains are summarized below inArea %.

P. krugani P. stagnora P. zopfii Fatty Acid (UTEX 329) (UTEX 1442) (UTEX1438) C 10:0 0.0 0.0 0.0 C 10:1 0.0 0.0 0.0 C 12:0 1.5 0.8 2.1 C 14:01.2 0.9 1.7 C 16 15.1 17.1 19.7 C 18:0 3.3 4.1 5.4 C 18:1 66.0 61.5 53.8C 18:2 12.9 15.6 17.3

These three strains were transformed with a yeast invertase (suc2)expression cassette (SEQ ID NO: 58) using the methods described inExample 3 above. This yeast invertase (suc2) expression cassette hasbeen demonstrated to work in Prototheca moriformis (UTEX 1435) above inExample 3. The transformants were screened using sucrose containingplates/media. A subset of the positive clones for each Protothecaspecies was selected and the presence of the transgene was confirmed bySouthern blot analysis. Ten of confirmed positive clones from eachspecies were selected for sucrose hydrolysis analysis and lipidproductivity. The clones were grown in media containing sucrose as thesole carbon source and compared to its wildtype counterpart grown onglucose. After 6 days, the cultures were harvested and dried and totalpercent lipid and dry cell weight was assessed. The media from eachculture was also analyzed for sucrose hydrolysis using a YSI2700Biochemistry Analyzer for sucrose content over the course of theexperiment. Clones from all three species were able to hydrolyzesucrose, with Prototheca stagnora and Prototheca zopfii transformantsbeing able to hydrolyze sucrose more efficiently than Protothecakrugani. Total lipid production and dry cell weight of the three speciesof transformants were comparable to their wildtype counterpart grown onglucose. This data demonstrates the successful transformation andexpression exogenous genes in multiple species of the genus Prototheca.

Example 11 Algal-Derived Promoters and Genes for Use in Microalgae

A. 5′UTR and Promoter Sequences from Chlorella protothecoides

A cDNA library was generated from mixotrophically grown Chlorellaprotothecoides (UTEX 250) using standard techniques. Based upon the cDNAsequences, primers were designed in certain known housekeeping genes to“walk” upstream of the coding regions using Seegene's DNA Walking kit(Rockville, Md.). Sequences isolated include an actin (SEQ ID NO:31) andelongation factor-1a (EF1a) (SEQ ID NO:32) promoter/UTR, both of whichcontain introns (as shown in the lower case) and exons (upper caseitalicized) and the predicted start site (in bold) and two beta-tubulinpromoter/UTR elements: Isoform A (SEQ ID NO:33) and Isoform B (SEQ IDNO:34).

B. Lipid Biosynthesis Enzyme and Plastid Targeting Sequences from C.protothecoides

From the cDNA library described above, three cDNAs encoding proteinsfunctional in lipid metabolism in Chlorella protothecoides (UTEX 250)were cloned using the same methods as described above. The nucleotideand amino acid sequences for an acyl ACP desaturase (SEQ ID NOs: 45 and46) and two geranyl geranyl diphosphate synthases (SEQ ID NOs:47-50) areincluded in the Sequence Listing below. Additionally, three cDNAs withputative signal sequences targeting to the plastid were also cloned. Thenucleotide and amino acid sequences for a glyceraldehyde-3-phosphatedehydrogenase (SEQ ID NOs:51 and 52), an oxygen evolving complex proteinOEE33 (SEQ ID NOs:53 and 54) and a Clp protease (SEQ ID NOs:55 and 56)are included in the Sequence Listing below. The putative plastidtargeting sequence has been underlined in both the nucleotide and aminoacid sequence. The plastid targeting sequences can be used to target theproducts of transgenes to the plastid of microbes, such as lipidmodification enzymes.

Example 12 5′UTR/Promoters that are Nitrogen Responsive from Protothecamoriformis

A cDNA library was generated from Prototheca moriformis (UTEX 1435)using standard techniques. The Prototheca moriformis cells were grownfor 48 hours under nitrogen replete conditions. Then a 5% innoculum(v/v) was then transferred to low nitrogen and the cells were harvestedevery 24 hours for seven days. After about 24 hours in culture, thenitrogen supply in the media was completely depleted. The collectedsamples were immediately frozen using dry ice and isopropanol. Total RNAwas subsequently isolated from the frozen cell pellet samples and aportion from each sample was held in reserve for RT-PCR studies. Therest of the total RNA harvested from the samples was subjected to polyAselection. Equimolar amounts of polyA selected RNA from each conditionwas then pooled and used to generate a cDNA library in vector pcDNA 3.0(Invitrogen). Roughly 1200 clones were randomly picked from theresulting pooled cDNA library and subjected to sequencing on bothstrands. Approximately 68 different cDNAs were selected from among these1200 sequences and used to design cDNA-specific primers for use inreal-time RT-PCR studies.

RNA isolated from the cell pellet samples that were held in reserve wasused as substrate in the real time RT-PCR studies using thecDNA-specific primer sets generated above. This reserved RNA wasconverted into cDNA and used as substrate for RT-PCR for each of the 68gene specific primer sets. Threshold cycle or C_(T) numbers were used toindicate relative transcript abundance for each of the 68 cDNAs withineach RNA sample collected throughout the time course. cDNAs showingsignificant increase (greater than three fold) between nitrogen repleteand nitrogen-depleted conditions were flagged as potential genes whoseexpression was up-regulated by nitrogen depletion. As discussed in thespecification, nitrogen depletion/limitation is a known inducer oflipogenesis in oleaginous microorganisms.

In order to identify putative promoters/5′UTR sequences from the cDNAswhose expression was upregulated during nitrogen depletion/limitation,total DNA was isolated from Prototheca moriformis (UTEX 1435) grownunder nitrogen replete conditions and were then subjected to sequencingusing 454 sequencing technology (Roche). cDNAs flagged as beingup-regulated by the RT-PCR results above were compared using BLASTagainst assembled contigs arising from the 454 genomic sequencing reads.The 5′ ends of cDNAs were mapped to specific contigs, and wherepossible, greater than 500 bp of 5′ flanking DNA was used to putativelyidentify promoters/UTRs. The presence of promoters/5′UTR weresubsequently confirmed and cloned using PCR amplification of genomicDNA. Individual cDNA 5′ ends were used to design 3′ primers and 5′ endof the 454 contig assemblies were used to design 5′ gene-specificprimers.

As a first screen, one of the putative promoter, the 5′UTR/promoterisolated from Aat2 (Ammonium transporter, SEQ ID NO: 99), was clonedinto the Cinnamomum camphora C14 thioesterase construct with theChlorella protothecoides stearoyl ACP desaturase transit peptidedescribed in Example 5 above, replacing the C. sorokinana glutamatedehydrogenase promoter. This construct is listed as SEQ ID NO: 112. Totest the putative promoter, the thioesterase construct is transformedinto Prototheca moriformis cells to confirm actual promoter activity byscreening for an increase in C14/C12 fatty acids under low/no nitrogenconditions, using the methods described above. Similar testing of theputative nitrogen-regulated promoters isolated from the cDNA/genomicscreen can be done using the same methods.

Other putative nitrogen-regulated promoters/5′UTRs that were isolatedfrom the cDNA/genomic screen were:

Promoter/5′UTR SEQ ID NO. Fold increased FatB/A promoter/5′UTR SEQ IDNO: 91 n/a NRAMP metal transporter promoter/5′UTR SEQ ID NO: 92 9.65Flap Flagellar-associated protein promoter/5′UTR SEQ ID NO: 93 4.92SulfRed Sulfite reductase promoter/5′UTR SEQ ID NO: 94 10.91 SugT Sugartransporter promoter/5′UTR SEQ ID NO: 95 17.35 Amt03-Ammoniumtransporter 03 promoter/5′UTR SEQ ID NO: 96 10.1 Amt02-Ammoniumtransporter 02 promoter/5′UTR SEQ ID NO: 97 10.76 Aat01-Amino acidtransporter 01 promoter/5′UTR SEQ ID NO: 98 6.21 Aat02-Amino acidtransporter 02 promoter/5′UTR SEQ ID NO: 99 6.5 Aat03-Amino acidtransporter 03 promoter/5′UTR SEQ ID NO: 100 7.87 Aat04-Amino acidtransporter 04 promoter/5′UTR SEQ ID NO: 101 10.95 Aat05-Amino acidtransporter 05 promoter/5′UTR SEQ ID NO: 102 6.71Fold increase refers to the fold increase in cDNA abundance after 24hours of culture in low nitrogen medium.

Example 13 Homologous Recombination in Prototheca Species

Homologous recombination of transgenes has several advantages over thetransformation methods described in the above Examples. First, theintroduction of transgenes without homologous recombination can beunpredictable because there is no control over the number of copies ofthe plasmid that gets introduced into the cell. Also, the introductionof transgenes without homologous recombination can be unstable becausethe plasmid may remain episomal and is lost over subsequent celldivisions. Another advantage of homologous recombination is the abilityto “knock-out” gene targets, introduce epitope tags, switch promoters ofendogenous genes and otherwise alter gene targets (e.g., theintroduction of point mutations.

Two vectors were constructed using a specific region of the Protothecamoriformis (UTEX 1435) genome, designated KE858. KE858 is a 1.3 kb,genomic fragment that encompasses part of the coding region for aprotein that shares homology with the transfer RNA (tRNA) family ofproteins. Southern blots have shown that the KE858 sequence is presentin a single copy in the Prototheca moriformis (UTEX 1435) genome. Thefirst type of vector that was constructed, designated SZ725 (SEQ ID NO:103), consisted of the entire 1.3 kb KE858 fragment cloned into a pUC19vector backbone that also contains the optimized yeast invertase (suc2)gene used in Example 3 above. The KE858 fragment contains an uniqueSnaB1 site that does not occur anywhere else in the targeting construct.The second type of vector that was constructed, designated SZ726 (SEQ IDNO: 126), consisted of the KE858 sequence that had been disrupted by theinsertion of the yeast invertase gene (suc2) at the SnaB1 site withinthe KE858 genomic sequence. The entire DNA fragment containing the KE858sequences flanking the yeast invertase gene can be excised from thevector backbone by digestion with EcoRI, which cuts at either end of theKE858 region.

Both vectors were used to direct homologous recombination of the yeastinvertase gene (suc2) into the corresponding KE858 region of thePrototheca moriformis (UTEX 1435) genome. The linear DNA ends homologousto the genomic region that was being targeted for homologousrecombination were exposed by digesting the vector construct SZ725 withSnaB1 and vector construct SZ726 with EcoRI. The digested vectorconstructs were then introduced into Prototheca moriformis culturesusing methods described above in Example 3. Transformants from eachvector construct were then selected using sucrose plates. Tenindependent, clonally pure transformants from each vector transformationwere analyzed for successful recombination of the yeast invertase geneinto the desired genomic location (using Southern blots) and fortransgene stability.

Southern blot analysis of the SZ725 transformants showed that 4 out ofthe 10 transformants picked for analysis contained the predictedrecombinant bands, indicating that a single crossover event had occurredbetween the KE858 sequences on the vector and the KE858 sequences in thegenome. In contrast, all ten of the SZ726 transformants contained thepredicted recombinant bands, indicating that double crossover events hadoccurred between the EcoRI fragment of pSZ726 carrying KE858 sequenceflanking the yeast invertase transgene and the corresponding KE858region of the genome.

Sucrose invertase expression and transgene stability were assessed bygrowing the transformants for over 15 generations in the absence ofselection. The four SZ725 transformants and the ten SZ276 transformantsthat were positive for the transgene by Southern blotting were selectedand 48 single colonies from each of the transformants were grownserially: first without selection in glucose containing media and thenwith selection in media containing sucrose as the sole carbon source.All ten SZ276 transformants (100%) retained their ability to grow onsucrose after 15 generations, whereas about 97% of the SZ725transformants retained their ability to grow on sucrose after 15generations. Transgenes introduced by a double crossover event (SZ726vector) have extremely high stability over generation doublings. Incontrast, transgenes introduced by a single cross over event (SZ725vector) can result in some instability over generation doublings becauseis tandem copies of the transgenes were introduced, the repeatedhomologous regions flanking the transgenes may recombine and excise thetransgenic DNA located between them.

These experiments demonstrate the successful use of homologousrecombination to generate Prototheca transformants containing aheterologous sucrose invertase gene that is stably integrated into thenuclear chromosomes of the organism. The success of the homologousrecombination enables other genomic alterations in Prototheca, includinggene deletions, point mutations and epitope tagging a desired geneproduct. These experiments also demonstrate the first documented systemfor homologous recombination in the nuclear genome of an eukaryoticmicroalgae.

A. Use of Homologous Recombination to Knock-Out an Endogenous Protothecamoriformis Gene

In the Prototheca moriformis cDNA/genomic screen described in Example 11above, an endogenous stearoyl ACP desaturase (SAPD) cDNA was identified.Stearoyl ACP desaturase enzymes are part of the lipid synthesis pathwayand they function to introduce double bonds into the fatty acyl chains.In some cases, it may be advantages to knock-out or reduce theexpression of lipid pathway enzymes in order to alter a fatty acidprofile. A homologous recombination construct was created to assesswhether the expression of an endogenous stearoyl ACP desaturase enzymecan be reduced (or knocked out) and if a corresponding reduction inunsaturated fatty acids can be observed in the lipid profile of the hostcell. An approximately 1.5 kb coding sequence of a stearoyl ACPdesaturase gene from Prototheca moriformis (UTEX 1435) was identifiedand cloned (SEQ ID NO: 104). The homologous recombination construct wasconstructed using 0.5 kb of the SAPD coding sequence at the 5′end (5′targeting site), followed by the Chlamydomonas reinhardtii β-tublinpromoter driving a codon-optimized yeast sucrose invertase suc2 genewith the Chlorella vulgaris 3′UTR. The rest (˜1 kb) of the Protothecamoriformis SAPD coding sequence was then inserted after the C. vulgaris3′UTR to make up the 3′ targeting site. The sequence for this homologousrecombination cassette is listed in SEQ ID NO: 105. As shown above, thesuccess-rate for integration of the homologous recombination cassetteinto the nuclear genome can be increased by linearizing the cassettebefore transforming the microalgae, leaving exposed ends. The homologousrecombination cassette targeting an endogenous SAPD enzyme in Protothecamoriformis is linearized and then transformed into the host cell(Prototheca moriformis, UTEX 1435). A successful integration willeliminate the endogenous SAPD enzyme coding region from the host genomevia a double reciprocal recombination event, while expression of thenewly inserted suc2 gene will be regulated by the C. reinhardtiiβ-tubulin promoter. The resulting clones can be screened usingplates/media containing sucrose as the sole carbon source. Clonescontaining a successful integration of the homologous recombinationcassette will have the ability to grow on sucrose as the sole carbonsource and changes in overall saturation of the fatty acids in the lipidprofile will serve as a secondary confirmation factor. Additionally,Southern blotting assays using a probe specific for the yeast sucroseinvertase suc2 gene and RT-PCR can also confirm the presence andexpression of the invertase gene in positive clones. As an alternative,the same construct without the β-tubulin promoter can be used to excisethe endogenous SAPD enzyme coding region. In this case, the newlyinserted yeast sucrose invertase suc2 gene will be regulated by theendogenous SAPD promoter/5′UTR.

Example 14 Fuel Production

A. Extraction of Oil from Microalgae Using an Expeller Press and a PressAid

Microalgal biomass containing 38% oil by DCW was dried using a drumdryer resulting in resulting moisture content of 5-5.5%. The biomass wasfed into a French L250 press. 30.4 kg (67 lbs.) of biomass was fedthrough the press and no oil was recovered. The same dried microbialbiomass combined with varying percentage of switchgrass as a press aidwas fed through the press. The combination of dried microbial biomassand 20% w/w switchgrass yielded the best overall percentage oilrecovery. The pressed cakes were then subjected to hexane extraction andthe final yield for the 20% switchgrass condition was 61.6% of the totalavailable oil (calculated by weight). Biomass with above 50% oil drycell weight did not require the use of a pressing aid such asswitchgrass in order to liberate oil.

B. Monosaccharide Composition of Delipidated Prototheca moriformisBiomass

Prototheca moriformis (UTEX 1435) was grown in conditions and nutrientmedia (with 4% glucose) as described in Example 45 above. The microalgalbiomass was then harvested and dried using a drum dryer. The dried algalbiomass was lysed and the oil extracted using an expeller press asdescribed in Example 44 above. The residual oil in the pressed biomasswas then solvent extracted using petroleum ether. Residual petroleumether was evaporated from the delipidated meal using a Rotovapor (BuchiLabortechnik AG, Switzerland). Glycosyl (monosaccharide) compositionanalysis was then performed on the delipidated meal using combined gaschromatography/mass spectrometry (GC/MS) of the per-O-trimethylsily(TMS) derivatives of the monosaccharide methyl glycosides produced fromthe sample by acidic methanolysis. A sample of delipidated meal wassubjected to methanolysis in 1M HCl in methanol at 80° C. forapproximately 20 hours, followed by re-N-acetylation with pyridine andacetic anhydride in methanol (for detection of amino sugars). Thesamples were then per-O-trimethylsiylated by treatment with Tri-Sil(Pierce) at 80° C. for 30 minutes (see methods in Merkle and Poppe(1994) Methods Enzymol. 230: 1-15 and York et al., (1985) MethodsEnzymol. 118:3-40). GC/MS analysis of the TMS methyl glycosides wasperformed on an HP 6890 GC interfaced to a 5975b MSD, using a All TechEC-1 fused silica capillary column (30 m×0.25 mm ID). Themonosaccharides were identified by their retention times in comparisonto standards, and the carbohydrate character of these are authenticatedby their mass spectra. 20 micrograms per sample of inositol was added tothe sample before derivatization as an internal standard. Themonosaccharide profile of the delipidated Prototheca moriformis (UTEX1435) biomass is summarized in Table 18 below. The total percentcarbohydrate from the sample was calculated to be 28.7%.

TABLE 18 Monosaccharide (glycosyl) composition analysis of Protothecamoriformis (UTEX 1435) delipidated biomass. Mole % (of total Mass (μg)carbohydrate) Arabinose 0.6 1.2 Xylose n.d. n.d. Galacturonic acid(GalUA) n.d. n.d. Mannose 6.9 11.9 Galactose 14.5 25.2 Glucose 35.5 61.7N Acetyl Galactosamine (GalNAc) n.d. n.d. N Acetyl Glucosamine (GlcNAc)n.d. n.d. Heptose n.d. n.d. 3 Deoxy-2-manno-2 Octulsonic n.d. n.d. acid(KDO) Sum 57 100 n.d. = none detected

The carbohydrate content and monosaccharide composition of thedelipidated meal makes it suitable for use as an animal feed or as partof an animal feed formulation. Thus, in one aspect, the presentinvention provides delipidated meal having the product content set forthin the table above.

C. Production of Biodiesel from Prototheca Oil

Degummed oil from Prototheca moriformis UTEX 1435, produced according tothe methods described above, was subjected to transesterification toproduce fatty acid methyl esters. Results are shown below:

The lipid profile of the oil was:

C10:0 0.02

C12:0 0.06

C14:0 1.81

C14.1 0.07

C16:0 24.53

C16:1 1.22

C18:0 2.34

C18:1 59.21

C18:2 8.91

C18:3 0.28

C20:0 0.23

C20:1 0.10

C20:1 0.08

C21:0 0.02

C22:0 0.06

C24:0 0.10

TABLE 19 Biodiesel profile from Prototheca moriformis triglyceride oil.Method Test Result Units ASTM Cold Soak Filterability of Filtration Time120 sec D6751 A1 Biodiesel Blend Fuels Volume Filtered 300 ml ASTM D93Pensky-Martens Closed Cup Procedure Used A Flash Point Corrected Flash165.0 ° C. Point ASTM Water and Sediment in Middle Sediment and Water0.000 Vol % D2709 Distillate Fuels (Centrifuge Method) EN 14538Determination of Ca and Mg Sum of (Ca and <1 mg/kg Content by ICP OESMg) EN 14538 Determination of Ca and Mg Sum of (Na and K) <1 mg/kgContent by ICP OES ASTM D445 Kinematic/Dynamic Kinematic Viscosity 4.873mm²/s Viscosity @ 104° F./40° C. ASTM D874 Sulfated Ash from LubricatingSulfated Ash <0.005 Wt % Oils and Additives ASTM Determination of TotalSulfur Sulfur, mg/kg 1.7 mg/kg D5453 in Light Hydrocarbons, SparkIgnition Engine Fuel, Diesel Engine Fuel, and Engine Oil by UltravioletFluorescence. ASTM D130 Corrosion - Copper Strip Biodiesel-Cu 1aCorrosion 50° C. (122° F.)/3 hr ASTM Cloud Point Cloud Point 6 ° C.D2500 ASTM Micro Carbon Residue Average Micro <0.10 Wt % D4530 MethodCarbon Residue ASTM D664 Acid Number of Petroleum Procedure Used AProducts by Potentiometric Acid Number 0.20 mg Titration KOH/g ASTMDetermination of Free and Free Glycerin <0.005 Wt % D6584 Total Glycerinin B-100 Total Glycerin 0.123 Wt % Biodiesel Methyl Esters By GasChromatography ASTM Additive Elements in Phosphorus 0.000200 Wt % D4951Lubricating Oils by ICP-AES ASTM Distillation of Petroleum IBP 248 ° C.D1160 Products at Reduced Pressure AET @ 5% 336 ° C. Recovery AET @ 10%338 ° C. Recovery AET @ 20% 339 ° C. Recovery AET @ 30% 340 ° C.Recovery AET @ 40% 342 ° C. Recovery AET @ 50% 344 ° C. Recovery AET @60% 345 ° C. Recovery AET @ 70% 347 ° C. Recovery AET @ 80% 349 ° C.Recovery AET @ 90% 351 ° C. Recovery AET @ 95% 353 ° C. Recovery FBP 362° C. % Recovered 98.5 % % Loss 1.5 % % Residue 0.0 % Cold Trap Volume0.0 ml IBP 248 ° C. EN 14112 Determination of Oxidation OxidationStability >12 hr Stability (Accelerated Operating Temp 110 ° C.Oxidation Test) (usually 110 deg C.) ASTM Density of Liquids by DigitalAPI Gravity @ 60° F. 29.5 °API D4052 Density Meter ASTM D6890Determination of Ignition Derived Cetane >61.0 Delay (ID) and DerivedNumber (DCN) Cetane Number (DCN)

The lipid profile of the biodiesel was highly similar to the lipidprofile of the feedstock oil. Other oils provided by the methods andcompositions of the invention can be subjected to transesterification toyield biodiesel with lipid profiles including (a) at least 4% C8-C14;(b) at least 0.3% C8; (c) at least 2% C10; (d) at least 2% C12; and (3)at least 30% C8-C14.

The Cold Soak Filterability by the ASTM D6751 A1 method of the biodieselproduced was 120 seconds for a volume of 300 ml. This test involvesfiltration of 300 ml of B100, chilled to 40° F. for 16 hours, allowed towarm to room temp, and filtered under vacuum using 0.7 micron glassfiber filter with stainless steel support. Oils of the invention can betransesterified to generate biodiesel with a cold soak time of less than120 seconds, less than 100 seconds, and less than 90 seconds.

D. Production of Renewable Diesel

Degummed oil from Prototheca moriformis UTEX 1435, produced according tothe methods described above and having the same lipid profile as the oilused to make biodiesel in Example X above, was subjected totransesterification to produce renewable diesel.

The oil was first hydrotreated to remove oxygen and the glycerolbackbone, yielding n-paraffins. The n-parrafins were then subjected tocracking and isomerization. A chromatogram of the material is shown inFIG. 13. The material was then subjected to cold filtration, whichremoved about 5% of the C18 material. Following the cold filtration thetotal volume material was cut to flash point and evaluated for flashpoint, ASTM D-86 distillation distribution, cloud point and viscosity.Flash point was 63° C.; viscosity was 2.86 cSt (centistokes); cloudpoint was 4° C. ASTM D86 distillation values are shown in Table 20:

TABLE 20 Readings in ° C.: Volume Temperature IBP 173  5 217.4 10 242.115 255.8 20 265.6 30 277.3 40 283.5 50 286.6 60 289.4 70 290.9 80 294.390 300 95 307.7 FBP 331.5

The T10-T90 of the material produced was 57.9° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10-T90 ranges, such as 20, 25,30, 35, 40, 45, 50, 60 and 65° C. using triglyceride oils producedaccording to the methods disclosed herein.

The T10 of the material produced was 242.1° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein, can be employed to generate renewablediesel compositions with other T10 values, such as T10 between 180 and295, between 190 and 270, between 210 and 250, between 225 and 245, andat least 290.

The T90 of the material produced was 300° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein can be employed to generate renewablediesel compositions with other T90 values, such as T90 between 280 and380, between 290 and 360, between 300 and 350, between 310 and 340, andat least 290.

The FBP of the material produced was 300° C. Methods of hydrotreating,isomerization, and other covalent modification of oils disclosed herein,as well as methods of distillation and fractionation (such as coldfiltration) disclosed herein, can be employed to generate renewablediesel compositions with other FBP values, such as FBP between 290 and400, between 300 and 385, between 310 and 370, between 315 and 360, andat least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 4% C8-C14; (b) at least 0.3% C8; (c) at least 2% C10; (d) atleast 2% C12; and (3) at least 30% C8-C14.

Example 15 Utilization of Sucrose by Chlorella luteoviridis

A. SAG 2214 Growth on Glucose and Sucrose

SAG 2214 (designated as Chlorella luteoviridis) was tested for growth inthe dark on media containing either glucose or sucrose. Heterotrophicliquid cultures were initiated using inoculum from a frozen vial ineither media containing 4% glucose or 4% sucrose as the sole carbonsource. Cultures were grown in the dark, shaking at 200 rpm. Samplesfrom the cultures were taken at 0, 24, 48 and 72 hour timepoints andgrowth was measured by relative absorbance at 750 nm (UV Mini1240,Shimadzu). SAG 2214 grew equally well on glucose as on sucrose, showingthat this microalgae can utilize sucrose as effectively as glucose as asole carbon source. The result of this experiment is representedgraphically in FIG. 3.

B. Lipid Productivity and Fatty Acid Profile for SAG 2214

Microalgal strain SAG 2214 was cultivated in liquid medium containingeither glucose or sucrose as the sole carbon source in similarconditions as described in Example 32 above. After 7 days, cells wereharvested for dry cell weight calculation. Cells were centrifuged andlyophilized for 24 hours. The dried cell pellets were weighed and thedry cell weight per liter was calculated. Cells for lipid analysis werealso harvested and centrifuged at 4000×g for 10 minutes at roomtemperature. The supernatant was discarded and the samples wereprocessed for lipid analysis and fatty acid profile using standard gaschromatography (GC/FID) procedures. The results are summarized below inTables 21 and 22.

TABLE 21 Lipid productivity and DCW for SAG 2214. DCW Sample Lipid (g/L)(g/L) % Lipid DCW SAG 2214 2.43 5.73 42.44% glucose SAG 2214 0.91 2.0045.56% sucrose

TABLE 22 Fatty acid profile for SAG 2214. Fatty Acid Percent (w/w)C:16:0 21 C:18:1 38 C:18:2 41

C. Genomic Comparison of SAG 2214 to Other Chlorella luteoviridisStrains

Microalgal strain SAG 2214 proved to be of general interest due to itsability to grow on sucrose as a carbon source (illustrated above). Inaddition to the growth characteristics of this strain, its taxonomicrelationship to other microalgal species was also of interest.Designated by the SAG collection as a Chlorella luteoviridis strain, the23s rRNA gene of SAG 2214 was sequenced and compared to the 23s rRNAgenomic sequence of nine other strains also identified by the SAG andUTEX collections as Chlorella luteoviridis. These strains were UTEX 21,22, 28, 257 and 258, and SAG strains 2133, 2196, 2198 and 2203. The DNAgenotyping methods used were the same as the methods described above inExample 1. Sequence alignments and unrooted trees were generated usingGeneious DNA analysis software. Out of the nine other strains that weregenotypes, UTEX 21, 22, 28 and 257 had identical 23s rRNA DNA sequence(SEQ ID NO: 106). The other five Chlorella luteoviridis strains had 23srRNA sequences that were highly homologous to UTEX 21, 22, 28, and 257.

The 23s rRNA gene sequence from SAG 2214 (SEQ ID NO: 30) is decidedlydifferent from that of the other nine C. luteoviridis strains, having alarge insertion that was not found in the other strains. Furtheranalysis of this 23s rRNA gene sequence using BLAST indicated that itshared the greatest homology with members of the genus Leptosira andTrebouxia (members of phycobiont portion of lichens). These resultsindicate that SAG 2214 may not be Chlorella luteoviridis strain ascategorized by the strain collection, but instead shares significant 23SrRNA nucleotide identity to algal symbionts found in lichen. The genomicanalysis along with the growth characteristics indicate that SAG 2214may be a source for genes and proteins involved in the metabolism ofsucrose, as well as signaling and transit peptides responsible for thecorrect localization of such enzymes. SAG 2214 and other strains with ahigh degree of genomic similarity may also be strains useful for oilproduction using sucrose as a source of fixed carbon.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein. In particular, the following patent applications arehereby incorporated by reference in their entireties for all purposes:U.S. Provisional Application No. 60/941,581, filed Jun. 1, 2007,entitled “Production of Hydrocarbons in Microorganisms”; U.S.Provisional Application No. 60/959,174, filed Jul. 10, 2007, entitled“Production of Hydrocarbons in Microorganisms”; U.S. ProvisionalApplication No. 60/968,291, filed Aug. 27, 2007, entitled “Production ofHydrocarbons in Microorganisms”; U.S. Provisional Application No.61/024,069, filed Jan. 28, 2008, entitled “Production of Hydrocarbons inMicroorganims”; PCT Application No. PCT/US08/65563, filed Jun. 2, 2008,entitled “Production of Oil in Microorganisms”; U.S. patent applicationSer. No. 12/131,783, filed Jun. 2, 2008, entitled “Use of CellulosicMaterial for Cultivation of Microorganisms”; U.S. patent applicationSer. No. 12/131,773, filed Jun. 2, 2008, entitled “Renewable Diesel andJet Fuel from Microbial Sources”; U.S. patent application Ser. No.12/131,793, filed Jun. 2, 2008, entitled “Sucrose Feedstock Utilizationfor Oil-Based Fuel Manufacturing”; U.S. patent application Ser. No.12/131,766, filed Jun. 2, 2008, entitled “Glycerol Feedstock Utilizationfor Oil-Based Fuel Manufacturing”; U.S. patent application Ser. No.12/131,804, filed Jun. 2, 2008, entitled “Lipid Pathway Modification inOil-Bearing Microorganisms”; U.S. Patent Application No. 61/118,590,filed Nov. 28, 2008, entitled “Production of Oil in Microorganisms”;U.S. Provisional Patent Application No. 61/118,994, filed Dec. 1, 2008,entitled “Production of Oil in Microorganisms”; U.S. Provisional PatentApplication No. 61/174,357, filed Apr. 3, 2009, entitled “Production ofOil in Microorganisms”; U.S. Provisional Patent Application No.61/219,525, filed Jun. 23, 2009, entitled “Production of Oil inMicroorganisms”; U.S. patent application Ser. No. 12/628,140, filed Nov.30, 2009, entitled “Novel Triglyceride and Fuel Compositions”; U.S.patent application Ser. No. 12/628,144, filed Nov. 30, 2009, entitled“Cellulosic Cultivation of Oleaginous Microorganisms”; U.S. patentapplication Ser. No. 12/628,147, filed Nov. 30, 2009, entitled “NucleicAcids Useful in the Manufacture of Oil”; U.S. patent application Ser.No. 12/628,149, filed Nov. 30, 2009, entitled, “Renewable ChemicalProduction from Novel Fatty Acid Feedstocks”, and U.S. patentapplication Ser. No. 12/628,120, filed Nov. 30, 2009, entitled“Recombinant Microalgae Cells Producing Novel Oils”.

What is claimed is:
 1. A polynucleotide comprising a first codingsequence and a second coding sequence, wherein the first coding sequenceencodes a fatty acyl-ACP thioesterase and the second coding sequenceencodes a sucrose invertase.
 2. The polynucleotide of claim 1, whereinthe second coding sequence encodes a yeast sucrose invertase or anArabidopsis invertase.
 3. The polynucleotide of claim 1, wherein thefirst coding sequence encodes a fatty acyl-ACP thioesterase selectedfrom the group consisting of the fatty acyl-ACP thioesterases listed inTable
 3. 4. The polynucleotide of claim 1, wherein the first codingsequence and the second coding sequence are codon-optimized forexpression in Prototheca spp.
 5. The polynucleotide of claim 1 furthercomprising a promoter sequence endogenous to a species of the genusPrototheca.
 6. The polynucleotide of claim 1, wherein the fatty acyl-ACPthioesterase has a preference for cleaving a fatty acid having 8-18carbon atoms.
 7. The polynucleotide of claim 1, wherein the fattyacyl-ACP thioesterase has a preference for cleaving a fatty acid having8-14 carbon atoms.
 8. The polynucleotide of claim 1, wherein the fattyacyl-ACP thioesterase has a preference for cleaving a fatty acid having12 carbon atoms.
 9. The polynucleotide of claim 1, wherein the secondcoding sequence encodes a sucrose invertase selected from the groupconsisting of the sucrose invertases listed in Table
 2. 10. Thepolynucleotide of claim 2, wherein the second coding sequence encodes ayeast sucrose invertase.
 11. The polynucleotide of claim 10, wherein theyeast is Saccharomyces cerevisiae.